CN116064405A - Cell model for screening and controlling biological clock core gene Bmal1 pharmacodynamic substances - Google Patents
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Abstract
The present invention provides a cell model for screening a pharmacodynamic substance regulating a core gene Bmal1 of a biological clock, which is obtained by infecting 293T cells and hepG2 cells with a lentivirus obtained by transfecting 293T cells with a recombinant plasmid containing a Bmal1 gene promoter and a luciferase reporter gene. According to the present disclosure, a cell model capable of screening a pharmacological substance for the regulatory biological clock core gene Bmal1 can be provided.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a cell model for screening and controlling a biological clock core gene Bmal1 pharmacodynamic substance.
Background
Circadian rhythms, i.e. biological oscillations with a period of about 24 hours, are maintained by a timing system determined by an innate gene, known as the molecular circadian spring system. Most biological processes in mammals have a circadian rhythm of about 24 hours. The circadian system is hierarchically organized into a plurality of oscillator networks. The coordination of the whole body circadian system involves the regulation of signals emanating from the supravisual nucleus (SCN), the central main pacemaker in front of the hypothalamus, and the auxiliary clocks in the external brain areas of the SCN and peripheral organs, which regulate the physiological and behavioral characteristics of the living body.
Because the biological clock has important physiological and clinical characteristics, the biological clock has important significance for developing small molecular medicine modulators. BMAL1 is one of the important core clock genes, controlled by the mechanism of ROR/REV-ERB response element (RORE) dependence, playing an important role in stabilizing the molecular biological clock cycle. In addition to circadian rhythms, recent studies have shown that Rev-erb. Alpha. Also is involved in regulating a variety of physiological processes, such as metabolism and energy homeostasis, inflammation, cardiovascular function and neurological function.
With the rapid development of economy and society, the prevalence of chronic diseases, such as obesity, diabetes, hypertension, gout, cardiovascular and cerebrovascular diseases, and tumors, and metabolic diseases has been remarkably increased by changes in resident diets and lifestyle. The development of small molecules against chronic metabolic diseases such as diabetes has shown great significance. Diabetes is a chronic disease, and our country is the most worldwide diabetic, and at present, research shows that clock gene expression disorder affects the occurrence and development of diabetes, and transcriptional regulation plays an important role in controlling circadian rhythm oscillation of clock gene expression. While the orphan nuclear receptor Rev-erbalpha (Rev-erb. Alpha.) is considered to be the primary regulator of biological clock, the expression of the primary regulator Bmal1 of mammalian circadian rhythm is inversely related to Rev-erb. Alpha. MRNA levels. Therefore, establishing a cell model for screening and regulating the biological clock core gene Bmal1 pharmacodynamic substances will have important significance for assisting in regulating the chronic metabolic diseases caused by circadian rhythm disorder and improving sleep.
Disclosure of Invention
The present disclosure has been made in view of the above-described conventional art, and an object thereof is to provide a cell model for screening a pharmacodynamic substance for controlling a core gene Bmal1 of a biological clock.
To this end, the present disclosure provides a cell model for screening for a pharmacodynamic substance that modulates a core gene Bmal1 of a biological clock, which is obtained by infecting 293T cells and hepG2 cells with a lentivirus obtained by cotransfecting 293T cells with a recombinant plasmid containing a Bmal1 gene promoter and a luciferase reporter gene.
In the present disclosure, lentiviruses are packaged with recombinant plasmids containing Bmal1 gene promoters and dual luciferase reporter genes, and a drug screening cell model is constructed, so that the cell model can be used for screening and controlling biological clock core gene Bmal1 pharmacophore or lead compounds thereof.
In addition, in the cell model related to the present disclosure, optionally, the recombinant plasmid is a ligation product of the Bmal1 gene promoter and the luciferase reporter gene. Thus, it is possible to determine whether the Bmal1 gene promoter normally promotes downstream gene expression (i.e., transcriptional activity of the Bmal1 promoter) by detecting the fluorescence value of luciferase and based on the fluorescence value.
In addition, in the cell model according to the present disclosure, optionally, the method for constructing the recombinant plasmid includes: designing an upstream primer and a downstream primer matched with the sequence of the Bmal1 gene promoter; performing PCR amplification on the sequence of the Bmal1 gene promoter by using the upstream primer and the downstream primer to obtain an amplification product; preparing a vector plasmid containing the luciferase reporter gene, and performing enzyme digestion on the vector plasmid; the amplification product was ligated to the digested vector plasmid using ligase. Thus, a recombinant plasmid in which the Bmal1 gene promoter and the luciferase reporter gene are linked can be obtained.
In addition, in the cell model according to the present disclosure, a sequence of-106 bp to 65bp among the sequences of the Bmal1 gene promoter may be optionally subjected to PCR amplification as a target sequence. Thus, gene expression can be promoted by the Bmal1 gene promoter.
In addition, in the cell model to which the present disclosure relates, optionally, the luciferase reporter is a dual luciferase reporter. Under the condition, the working condition of the Bmal1 gene promoter can be judged through the fluorescence value of the double-luciferase reporter gene, so that experimental errors caused by experimental transfection operation are avoided, and the verification accuracy is improved.
Additionally, in the cell model to which the present disclosure relates, optionally, the luciferase reporter gene includes one or more of a firefly luciferase reporter gene and a sea cucumber luciferase reporter gene. Thus, the detection of the fluorescence value of the luciferase reporter gene can be facilitated.
In addition, in the cell model related to the disclosure, optionally, the nucleic acid sequence of the recombinant plasmid is shown as SEQ ID No. 1.
In addition, in the cell model to which the present disclosure relates, optionally, in the construction of the recombinant plasmid, a AAGTAGGTTAAAGTAGGTCA (SEQ ID NO. 2) wild-type sequence and a AAGTACATTAAAGTACATCA (SEQ ID NO. 3) mutant sequence are inserted in the multiple cloning site region. In this case, when screening a drug substance for controlling the core gene Bmal1 of the biological clock, if the drug contains a sequence matching the wild-type sequence, the sequence suppresses the expression of the downstream gene, and the fluorescence value detected at this time becomes low; if the medicine contains a sequence matched with the sequence containing the mutant, the downstream gene can be expressed normally, and the detected fluorescence value is unchanged, so that the medicine effect substance of the regulatory biological clock core gene Bmal1 can be screened.
In addition, in the cell model to which the present disclosure relates, optionally, the lentivirus is obtained by co-transfecting 293T cells with the recombinant plasmid and a lentiviral vector.
In addition, in the cell model related to the present disclosure, optionally, the method for constructing a lentiviral vector includes: preparing a lentiviral expression vector pLVX-Puro, and performing enzyme digestion on the lentiviral expression vector pLVX-Puro; preparing a first reporter gene pGL4.22[ luc2CP/Puro ] and an internal reference reporter gene pRL-TK, carrying out PCR amplification on the first reporter gene pGL4.22[ luc2CP/Puro ] to obtain a first amplification product, and carrying out PCR amplification on the internal reference reporter gene pRL-TK to obtain a second amplification product; and connecting the digested lentiviral expression vector pLVX-Puro with the first amplification product and the second amplification product to obtain the lentiviral vector. In this case, a cell line having a resistance gene can be selected by the first reporter gene, and the influence of an interference factor can be reduced by the reference reporter gene. Thus, lentiviral vectors for drug screening can be obtained.
According to the present disclosure, a cell model capable of screening a pharmacological substance for the regulatory biological clock core gene Bmal1 can be provided.
Drawings
Fig. 1 is a schematic diagram illustrating a map of a first reporter gene according to an embodiment of the present disclosure.
Fig. 2 is a schematic diagram showing recombinant plasmids according to embodiments of the present disclosure.
Fig. 3 is a schematic diagram showing lentiviral plasmids according to embodiments of the present disclosure.
FIG. 4 is a fluorescent diagram showing construction of a cell model for RORE-WT lentiviral-infected 293T cell line according to an embodiment of the present disclosure.
Figure 5 is a graph showing the results of dry prognosis bifluorescein reporting using different concentrations of NR1D1 agonist SR9009 for 293T cell lines according to embodiments of the present disclosure.
Fig. 6 is a graph showing the results of dry prognosis bifluorescein reporting using different concentrations of the NR1D1 inhibitor SR8278 for 293T cell lines according to embodiments of the present disclosure.
Fig. 7 is a graph showing dry prognosis bifluorescein reporting results using different concentrations of the agonist SR9009+sr8278 of NR1D1 for 293T cell lines according to embodiments of the present disclosure.
FIG. 8 is a fluorescent chart showing viral infection of hepG2 cell line according to embodiments of the present disclosure.
FIG. 9 is a graph showing the results of dry prognosis bifluorescein reporting using NR1D1 agonist SR9009 at different concentrations for hepG2 cell lines according to the examples of the present disclosure.
FIG. 10 is a graph showing the results of a dry prognosis dual fluorescein report for hepG2 cell lines according to the examples of the present disclosure using different concentrations of NR1D1 inhibitor SR 8278.
FIG. 11 is a graph showing the results of dry prognosis bifluorescein reporting using different concentrations of NR1D1 agonist SR9009+SR8278 for hepG2 cell lines according to the examples of the present disclosure.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements throughout or elements having like or similar functionality. The embodiments described below by way of the drawings are exemplary only and should not be construed as limiting the invention.
It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the prior art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
In order that the invention may be readily understood, a further description of the invention will be rendered by reference to specific embodiments that are illustrated in the appended drawings and are not to be construed as limiting embodiments of the invention. It will be appreciated by those skilled in the art that the drawings are merely schematic representations of examples and that the elements of the drawings are not necessarily required to practice the invention.
The present disclosure relates to a cell model for screening and controlling a biological clock core gene Bmal1 pharmacodynamic substance. Through the cell model disclosed by the invention, the drug effect substance of the control biological clock core gene Bmal1 or the lead compound thereof can be screened. For example, antidiabetic agents can be obtained by screening. In this disclosure, the cell model may also be referred to as a "drug screening cell model". In the present disclosure, a cell model for screening a drug substance regulating the biological clock core gene Bmal1 may also be referred to as a "drug screening cell model targeting the biological clock core gene Bmal1 promoter".
Hereinafter, a cell model according to the present disclosure will be described with reference to the accompanying drawings.
In this embodiment, the cell model can be obtained by infecting 293T cells and hepG2 cells with lentiviruses. Among them, lentiviruses can be obtained by cotransfecting 293T cells with a recombinant plasmid containing Bmal1 gene promoter and luciferase reporter gene. In this case, lentiviruses are packaged with recombinant plasmids containing Bmal1 gene promoter and dual luciferase reporter gene, and 293T cells and hepG2 cell models for drug screening are constructed, so that the cell models can be used for screening and controlling biological clock core gene Bmal1 pharmacophore or lead compounds thereof.
In some examples, the recombinant plasmid may be obtained by ligating a Bmal1 gene promoter with a luciferase reporter gene. In this case, it is possible to determine whether the Bmal1 gene promoter normally promotes the downstream gene expression (i.e., the transcriptional activity of the Bmal1 promoter) by detecting the fluorescence value of luciferase and based on the fluorescence value. Specifically, when the fluorescence value of luciferase in the recombinant plasmid is smaller than that of the control group (control plasmid containing no Bmal1 promoter), the transcriptional activity of the Bmal1 promoter is activated (i.e., the downstream gene expression can be normally promoted), and at this time, the Bmal1 promoter inhibits the gene expression of luciferase to affect the fluorescence value thereof.
In some examples, the recombinant plasmid may be obtained by: designing an upstream primer and a downstream primer matched with the sequence of the Bmal1 gene promoter (step S100); performing PCR amplification on the sequence of the Bmal1 gene promoter by using an upstream primer and a downstream primer to obtain an amplification product (step S200); preparing a vector plasmid containing a luciferase reporter gene, and performing enzyme digestion on the vector plasmid (step S300); the amplified product was ligated with the digested vector plasmid using T4 ligase (step S400). In this case, a recombinant plasmid in which the Bmal1 gene promoter and the luciferase reporter gene are ligated can be obtained.
In some examples, in step S200, the sequence of the upstream primer may be as shown in SEQ ID No. 4.
In some examples, in step S200, the sequence of the downstream primer may be as shown in SEQ ID No. 5.
In some examples, in step S300, the vector plasmid may be digested with KpnI and HindIII endonucleases.
In some examples, in step S300, the luciferase reporter may be a dual luciferase reporter. In this case, the operation of the Bmal1 gene promoter can be determined by the fluorescence value of the dual-luciferase reporter gene, thereby improving the verification accuracy.
In some examples, in step S300, the luciferase reporter may be one or more of a firefly luciferase reporter and a sea cucumber luciferase reporter. Thus, the detection of the fluorescence value of the luciferase reporter gene can be facilitated.
In some examples, in step S400, the ligase may be a T4 ligase.
In some examples, in step S400, the ligation product of the amplification product and the digested vector plasmid may be identified to determine whether the ligation product includes a sequence having a Bmal1 promoter. Specifically, the ligation product may be transformed, plated, and sequenced to yield the identification result.
In some examples, the recombinant plasmid may be a recombinant lentiviral plasmid.
In some examples, the nucleic acid sequence of the recombinant plasmid may be as shown in SEQ ID No. 1.
In some examples, recombinant plasmid construction may insert wild-type sequences and mutant sequences in the region of the multiple cloning site. In this case, when screening a drug substance for controlling the core gene Bmal1 of the biological clock, if the drug contains a sequence matching the wild-type sequence, the sequence suppresses the expression of the downstream gene, and the fluorescence value detected at this time becomes low; if the medicine contains a sequence matched with the sequence containing the mutant, the downstream gene can be expressed normally, and the detected fluorescence value is unchanged, so that the small molecule regulator for assisting in regulating the circadian rhythm and metabolism can be screened.
In some examples, the nucleic acid sequence of the wild-type sequence may be as set forth in SEQ ID No. 2: AAGTAGGTTAAAGTAGGTCA.
In some examples, the nucleic acid sequence of the mutant sequence may be as set forth in SEQ ID No. 3: AAGTACATTAAAGTACATCA.
In some examples, lentiviruses may be obtained by co-transfecting 293T cells with a recombinant plasmid and a lentiviral vector.
In some examples, the vector may be the lentivirus common vector pLVX-Puro.
In some examples, the method of constructing a lentiviral vector comprises: preparing a lentiviral expression vector pLVX-Puro, and performing enzyme digestion on the lentiviral expression vector pLVX-Puro; preparing a first reporter gene pGL4.22[ luc2CP/Puro ] and an internal reference reporter gene pRL-TK, carrying out PCR amplification on the first reporter gene pGL4.22[ luc2CP/Puro ] to obtain a first amplification product, and carrying out PCR amplification on the internal reference reporter gene pRL-TK to obtain a second amplification product; and connecting the digested lentiviral expression vector pLVX-Puro with the first amplification product and the second amplification product to obtain the lentiviral vector. In this case, a cell line having a resistance gene can be selected by the first reporter gene, and the influence of an interference factor can be reduced by the reference reporter gene. Thus, lentiviral vectors for drug screening can be obtained.
In some examples, the sequence of pGL4.22[ luc2CP/Puro ] -F may be as shown in SEQ ID No. 6: 5'-gcagagatccagtttatcgatGGCCTAACTGGCCGGTACC-3'.
In some examples, the sequence of pGL4.22[ luc2CP/Puro ] -R may be as shown in SEQ ID No. 7: 5'-gtactctagccttaAACTTGTTTATTGCAGCTTATAATGGT-3'.
In some examples, the sequence of pRL-TK-F may be set forth in SEQ ID No. 8: 5' -caagttTAAGGCTAGAGTACTTAATACGACTCACTATAG-3.
In some examples, the sequence of pRL-TK-R may be as shown in SEQ ID No. 9: 5'-ttatctagagtcgcgggatccAACTTGTTTATTGCAGCTTATAATGGT-3'.
In some examples, the method of constructing a cell model includes: constructing a recombinant plasmid containing a Bmal1 gene promoter and a luciferase reporter gene; constructing a lentiviral vector; co-transfecting 293T cells with the recombinant plasmid and the lentiviral vector to obtain lentivirus; cell models were obtained by infecting 293T cells and hepG2 cells with lentiviruses. Thus, a cell model which can screen the pharmacological substances of the regulatory biological clock core gene Bmal1 can be obtained.
In view of the above, in the present disclosure, a cell model capable of screening a pharmacological substance regulating the core gene Bmal1 of a biological clock can be provided.
The cell model provided by the present disclosure is described in detail below in connection with examples, but they should not be construed as limiting the scope of the present disclosure.
Examples (example)
Step one: construction of recombinant plasmids
In this example, a stepwise cloning program was used to construct the plasmid sequence of interest.
First, the MCS position of pGL4.22[ luc2CP/Puro ]. Dnase sequence to SV40ploy (A) (-106 bp to 65 bp) sequence was inserted in front of pRL-TK vector HSVTK promoter to construct an empty vector. Then, a wild-type sequence of AAGTAGGTTAAAGTAGGTCA (SEQ ID NO. 2) and a mutant sequence of AAGTACATTAAAGTACATCA (SEQ ID NO. 3) were inserted into the MCS region to construct two plasmids.
1. Designing primers
The Bmal1 promoter sequence was searched in NCBI database and its known literature, and primers for the Bmal1 promoter were designed by primer premier5.0 software. Using the human genome as a template, the upstream primer was 5'-cAAGTAGGTTAAAGTAGGTCAa-3' (SEQ ID NO. 4) and the downstream primer was 5'-AGCTTTGACCTACTTTAACCTACTTGGTAC-3' (SEQ ID NO. 5).
2. Primer annealing
The annealing system is Oligo-F (100 uM) 1ul, oligo-R (100 uM) 1ul, T4-PNK 1ul, buffer 1ul, ddH2O6ul, human cDNA template 2ul,95℃for 5min, 55℃for 15s, 72℃for 30s,72℃for 5min; reducing the temperature to 25 ℃ and reducing the temperature to 0.1 ℃ per second; 25 ℃ for 5min; 5min at 4 ℃; stored at 4 degrees.
pGL4.22[ luc2CP/Puro ] cleavage
The pGL4.22[ luc2CP/Puro ] plasmid was subjected to a double digestion experiment, the digestion reaction system was pGL4.22[ luc2CP/Puro ] plasmid 1ug, kp nI endonuclease 1ul, hindIII endonuclease 1ul,10 XBuffer Buffer 10ul, and ddH2O was added to prepare 50ul of the total volume of the digestion reaction system. And (3) performing enzyme digestion for 2-4 hours at 37 ℃ to obtain the linearized pGL4.22[ luc2CP/Puro ] plasmid. FIG. 1 is a schematic diagram showing a first reporter gene pGL4.22[ luc2CP/Puro ] according to embodiments of the present disclosure. The first reporter gene pGL4.22[ luc2CP/Puro ] contains a Puromycin resistance gene and Puromycin can be used to screen cells labeled with Puromycin resistance gene during cell model screening.
4. Connection
The Bmal1 promoter fragment was ligated to the linearized pGL4.22[ luc2CP/Puro ] plasmid in a manner that the ligation system was 100ng of the linearized pGL4.22[ luc2CP/Puro ] plasmid, 33ug of the insert, 1ul of T4 ligase, 1ul of buffer, and the total volume of the ligation reaction system was made 10ul by adding ddH 2O. Ligation was performed at 25℃for 3 hours to obtain a ligation product, i.e.the recombinant plasmid pRL-TK-master-Bmal 1-Luc2CP. FIG. 2 is a schematic diagram showing recombinant plasmid pRL-TK-Promoter-Bmal1-Luc2CP according to the examples of the present disclosure. The recombinant plasmid pRL-TK-Promoter-Bmal1-Luc2CP contains a Bmal1 gene Promoter sequence, and can be used for screening and controlling biological clock core gene Bmal1 pharmacodynamic substances by using a cell model constructed by the recombinant plasmid pRL-TK-Promoter-Bmal1-Luc2CP.
5. Sequencing after conversion plating
And (3) carrying out transformation plating and sequencing on the connection product, wherein the sequencing result is consistent with the target sequence.
Step two: construction of lentiviral vectors
pLVX-Puro cleavage
The lentiviral expression vector pLVX-Puro is digested, the digestion reaction system is 1ug of pLVX-Puro vector, 1ul of ClaI endonuclease, 1ul of BamHI endonuclease and 10ul of 10 XBuffer Buffer, and ddH2O is added to make the total volume of the digestion reaction system 50ul. And performing enzyme digestion for 2-4h at 37 ℃.
PCR amplification
Designing primers
PCR amplification
PCR reaction procedure
3. Connection
The inserts were ligated to lentiviral expression vectors in a ligation system of linearized pLVX-Puro vector 162ng, puro-RORE (WT) -hFluc with 2 inserts (n.ltoreq.5) of SV 40-promter-hRluc (23ng+42 ng,2 XClonExpress Mix2 ul) and ddH2O was added to make the total ligation system volume 10ul. The lentiviral plasmid was obtained by cooling to 50℃for 15 min, down to 4℃or immediately cooling on ice. Fig. 3 is a schematic diagram showing lentiviral plasmids according to embodiments of the present disclosure. The Bmal1 gene promoter sequence and the luciferase reporter gene can be stably integrated on the genome of 293T cells and hepG2 cells by transfecting the cells with lentiviral plasmids, so as to construct a stable cell model.
4. Sequencing
Sequencing the recombinant lentiviral plasmid, and confirming that the sequence of the recombinant lentiviral plasmid is completely correct according to the sequencing result.
Step three: lentivirus package and medicine cell model obtained by infecting cell line
(1) The first day: preparation of cells
Well conditioned 293T cells and hepG2 cells were seeded into 24 well cell culture plates.
After cell digestion, counting, adjusting the density of the cell suspension to 1X 104< cells/mL, adding 500 mu l of the cell suspension into each hole for plating, and plating at the density of 5-10X 103 cells/hole.
(2) The following day: viral infection
1) Calculating virus sample addition amount
The virus infection is performed by selecting the appropriate MOI and the infection conditions.
The sample adding amount calculating method comprises the following steps: (cell number. Times. MOI/viral stock titer). Times.103 = viral load (. Mu.L), e.g., cell number 5. Times.104, MOI 20, viral titer 5. Times.108 TU/mL, viral load 2. Mu.L.
2) Infection with
Observing that 293T cells and hepG2 cells are in good state, and adding lentivirus for infection when the cell density reaches 30-40%.
The medium in the well plate was removed, replaced with fresh cell medium, and the corresponding amount of virus was added.
(3) Third day: liquid exchange
After 12-16 hours of virus infection, observing the cell state and replacing the fresh culture medium. The medium in the well plate was removed and 500uL of complete medium was added per well. If the cell state is bad, the fresh culture medium needs to be replaced as soon as possible; if the cell state is good, the liquid change can be performed within 24 hours, but the liquid change is not preferable to be performed for more than 24 hours.
(4) Fifth day: observation conditions
After 48 hours post infection, the virus carrying the Puromycin resistance gene was replaced with fresh complete medium containing appropriate concentrations of Puromycin and stably transduced cell lines were screened.
(5) Puromycin resistance screening:
puromycin was applied at a final concentration of 5. Mu.g/ml, and an equal amount of Puromycin was added to a non-infectious screening control (wild type cells not infected with virus).
Co-transfecting a recombinant plasmid pRL-TK-Promoter-Bmal1-Luc2CP carrying a target sequence with a lentiviral vector to obtain lentivirus by a 293T cell line prepared in advance, and judging the transcriptional activity of a Bmal1 Promoter by detecting the fluorescence value of luciferase; the lentivirus was then infected with 293T and hepG2 cell lines and the transcriptional activity of the Bmal1 promoter was determined by detecting the fluorescent value of luciferase. FIG. 4 is a fluorescent diagram showing construction of a cell model for RORE-WT lentiviral-infected 293T cell line according to an embodiment of the present disclosure. FIG. 8 is a fluorescent chart showing viral infection of hepG2 cell line according to embodiments of the present disclosure. After lentivirus infection of 293T and hepG2 cell lines, fluorescence was observed by fluorescence microscopy, indicating successful integration of Bmal1 gene promoter sequences into the 293T and hepG2 cell genomes.
The constructed 293T stable screening cell line is subjected to interference screening through different concentrations of NR1D1 agonist SR9009, inhibitor SR8278 and R9009+SR8278 for different time periods of 0h, 24h and 48 h; and (3) performing interference screening on the constructed hepG2 stable screening cell line through different concentrations of NR1D1 agonist SR9009, inhibitor SR8278 and R9009+SR8278 for different time periods of 0h, 24h and 48h stimulation to obtain a drug cell model.
The fluorescence intensities of 293T cells and hepG2 cells after lentivirus infection are detected by a chemiluminescent instrument, and the relative luciferase activity is calculated and obtained, wherein the luciferase activity is in direct proportion to the transcriptional regulation activity of the Bmal1 gene promoter.
Figure 5 is a graph showing the results of dry prognosis bifluorescein reporting using different concentrations of NR1D1 agonist SR9009 for 293T cell lines according to embodiments of the present disclosure. Interference by stimulating lentiviral-infected 293T cells with different concentrations of NR1D1 agonist SR9009, and detecting the fluorescence intensities of 0h, 24h and 48h by a chemiluminescent instrument, shows that the fluorescence intensities of 24h and 48h decrease with increasing concentrations of the agonist SR9009, and shows that the transcriptional regulation activity of Bmal1 gene promoters at 24h and 48h decreases with increasing concentrations of the agonist SR 9009.
Fig. 6 is a graph showing the results of dry prognosis bifluorescein reporting using different concentrations of the NR1D1 inhibitor SR8278 for 293T cell lines according to embodiments of the present disclosure. Different concentrations of the NR1D1 inhibitor SR8278 were used to stimulate the intervention of lentivirally infected 293T cells. The fluorescence intensities of 0h, 24h and 48h were measured by a chemiluminescent apparatus, and the results showed that the fluorescence intensities of 24h and 48h tended to increase and then decrease with increasing concentration of the inhibitor SR8278, indicating that the transcriptional regulation activity of the Bmal1 gene promoters at 24h and 48h tended to increase and then decrease with increasing concentration of the inhibitor SR 8278.
Fig. 7 is a graph showing dry prognosis bifluorescein reporting results using different concentrations of the agonist SR9009+sr8278 of NR1D1 for 293T cell lines according to embodiments of the present disclosure. The results showed that the fluorescence intensities at 24h and 48h tended to increase and then remain substantially unchanged with increasing concentrations of the agonists SR9009+SR8278, indicating that the transcriptional regulatory activity of the Bmal1 gene promoter at 24h and 48h tended to increase and then remain unchanged with increasing concentrations of the agonists SR9009+SR 8278.
FIG. 9 is a graph showing the results of dry prognosis bifluorescein reporting using NR1D1 agonist SR9009 at different concentrations for hepG2 cell lines according to the examples of the present disclosure. Intervention with different concentrations of NR1D1 agonist SR9009 stimulated lentiviral infected hepG2 cells, and detection of 0h, 24h, 48h fluorescence intensities by chemiluminescence apparatus showed that the 24h and 48h fluorescence intensities decreased with increasing concentrations of agonist SR9009, indicating that transcriptional regulation activity of Bmal1 gene promoters at 24h and 48h decreased with increasing concentrations of agonist SR 9009.
FIG. 10 is a graph showing the results of a dry prognosis dual fluorescein report for hepG2 cell lines according to the examples of the present disclosure using different concentrations of NR1D1 inhibitor SR 8278. The interference of the lentiviral infected hepG2 cells is stimulated by adopting NR1D1 inhibitors SR8278 with different concentrations, and the fluorescence intensities of 0h, 24h and 48h are detected by a chemiluminescent instrument, so that the results show that the fluorescence intensities of 24h and 48h have a trend of increasing and then decreasing with the increasing concentration of the inhibitor SR8278, and the transcriptional regulation activity of Bmal1 gene promoters at 24h and 48h has a trend of increasing and then decreasing with the increasing concentration of the inhibitor SR 8278.
FIG. 11 is a graph showing the results of dry prognosis bifluorescein reporting using different concentrations of NR1D1 agonist SR9009+SR8278 for hepG2 cell lines according to the examples of the present disclosure. The results show that the fluorescence intensities of 24h and 48h decrease first and then increase and then decrease with increasing concentration of the agonist SR9009+SR8278, which indicates that the transcriptional regulation activity of the Bmal1 gene promoter at 24h and 48h decreases first and then increases and then decreases with increasing concentration of the agonist SR 9009+SR8278.
Thus, the cell model disclosed by the invention can be used for screening the drug effect substances of the control biological clock core gene Bmal 1.
While the disclosure has been described in detail in connection with the drawings and embodiments, it should be understood that the foregoing description is not intended to limit the disclosure in any way. Modifications and variations of the present disclosure may be made as desired by those skilled in the art without departing from the true spirit and scope of the disclosure, and such modifications and variations fall within the scope of the disclosure.
Claims (10)
1. A cell model for screening a pharmacodynamic substance for regulating a core gene Bmal1 of a biological clock, which is obtained by infecting 293T cells and hepG2 cells with a lentivirus obtained by transfecting 293T cells with a recombinant plasmid containing a Bmal1 gene promoter and a luciferase reporter gene.
2. The cell model of claim 1, wherein the recombinant plasmid is a ligation product of the Bmal1 gene promoter and the luciferase reporter gene.
3. The cell model of claim 2, wherein the construction method of the recombinant plasmid comprises: designing an upstream primer and a downstream primer matched with the sequence of the Bmal1 gene promoter; performing PCR amplification on the sequence of the Bmal1 gene promoter by using the upstream primer and the downstream primer to obtain an amplification product; preparing a vector plasmid containing the luciferase reporter gene, and performing enzyme digestion on the vector plasmid; the amplification product was ligated to the digested vector plasmid using ligase.
4. A cell model according to claim 3, wherein the sequence of-106 bp to 65bp of the sequence of the Bmal1 gene promoter is used as a target sequence for PCR amplification.
5. The cell model of claim 1, wherein the luciferase reporter is a dual luciferase reporter.
6. The cell model of claim 1, wherein the luciferase reporter gene comprises one or more of firefly luciferase reporter gene, sea cucumber luciferase reporter gene.
7. The cell model of claim 1, wherein the core key nucleic acid sequence of the recombinant plasmid is shown in SEQ ID No. 1.
8. The cell model of claim 1, wherein the recombinant plasmid is constructed by inserting a AAGTAGGTTAAAGTAGGTCA wild-type sequence and a AAGTACATTAAAGTACATCA mutant sequence into the region of the multiple cloning site.
9. The cell model of claim 1, wherein the lentivirus is obtained by co-transfecting 293T cells with the recombinant plasmid and a lentiviral vector.
10. The cell model of claim 9, wherein the method for constructing the lentiviral vector comprises: preparing a lentiviral expression vector pLVX-Puro, and performing enzyme digestion on the lentiviral expression vector pLVX-Puro; preparing a first reporter gene pGL4.22[ luc2CP/Puro ] and an internal reference reporter gene pRL-TK, carrying out PCR amplification on the first reporter gene pGL4.22[ luc2CP/Puro ] to obtain a first amplification product, and carrying out PCR amplification on the internal reference reporter gene pRL-TK to obtain a second amplification product; and connecting the digested lentiviral expression vector pLVX-Puro with the first amplification product and the second amplification product to obtain the lentiviral vector.
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