Method for reducing background and increasing Luciferase gene expression activity
By introducing specific nucleotide sequences and constructing the enhancer plasmid vector PmirGLO-Enhancer, the problem of low expression of Luciferase gene in some cells was solved, and the effect of significantly improving the expression activity of Luciferase gene was achieved.
Patent Information
- Application Number
- CN202311414098.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-10-27
- Publication Date
- 2025-05-06
AI Technical Summary
In the prior art, the firefly luciferase vector cannot maintain high expression on some cells, and the Basic plasmid vector still has a high expression background in cells and animals, resulting in unsatisfactory Luciferase gene expression.
By introducing specific nucleotide sequences, the enhancer plasmid vector PmirGLO-Enhancer was constructed, and cloned into the Luciferase gene upstream of the Luciferase gene promoter by PCR amplification and enzyme cleavage site BglII/ApaI, thereby enhancing the expression of the Luciferase gene.
It effectively reduces the background expression of the Luciferase gene, significantly increases its expression activity, and improves the expression efficiency in cells and animals.
Smart Images
Figure CN119932067A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for reducing background and increasing Luciferase gene expression activity. Background Art
[0002] Luciferase: refers to a class of enzymes that can catalyze the oxidation of different substrates (such as luciferin, coelenterazine, etc.) to emit fluorescence. The most representative luciferase is firefly luciferase (FL), which can catalyze the oxidation of luciferin to oxyluciferin, and emit fluorescence that can be detected by instruments during the process. Because of its easy operation, high sensitivity, small error, and wide applicability, it has become the most commonly used reporter gene in experiments. In addition, the renilla luciferase gene (Rluc), as another new type of reporter gene, has higher sensitivity and a wider linear range than firefly luciferase, so it is often used as an internal reference gene and firefly luciferase to form a dual luciferase reporter system.
[0003] Enhancer: It is a non-coding sequence in the genome, usually 100-1000bp in length. As a cis-regulatory element, enhancer is composed of dense clusters of transcription factor binding sites (TFBS), which can bind to proteins (such as specific transcription factors) and then recruit several types of protein complexes, including mediator complexes, histone modifiers, chromatin modifiers and structural proteins, to activate the expression of target genes transcribed by RNA polymerase II (RNAPII). At the same time, it can be independent of the direction, distance and position of the target gene. Enhancers regulate gene transcription by enhancing the accessibility of transcription start points, increasing the recruitment of RNA polymerase and enhancing the transcription rate, and are key elements in coordinating gene expression regulation.
[0004] In routine experiments, the commonly used firefly luciferase vector cannot maintain high expression in some cells, and the existing Basic plasmid vector (no promoter in front of the Luciferase gene) used as a negative control still has a high expression background in cells and animals. Therefore, it is necessary to find a method that can reduce the background and effectively increase the expression activity of the Luciferase gene. Summary of the invention
[0005] In order to solve the above-mentioned problems, the present invention discloses a nucleotide sequence and its application.
[0006] The present invention requires obtaining a specific nucleotide sequence: the nucleotide sequence is shown in SEQ ID No.1.
[0007] The primer pair required for amplifying the above-mentioned specific nucleotide sequence, the nucleotide sequence of the primer pair is shown in SEQ ID No. 2 and SEQ ID No. 3. The method for amplifying the above-mentioned specific nucleotide sequence is to use the synthetic DNA as a template and perform PCR amplification with the primer pair described in SEQ ID No. 2 and SEQ ID No. 3 to obtain the nucleotide sequence shown in SEQ ID No. 1.
[0008] Furthermore, the application of the above nucleotide sequence includes the following steps: before introducing the specific nucleotide sequence into the vector plasmid promoter, amplifying it through transformation by engineering bacteria, and finally transfecting it into cells for expression.
[0009] Furthermore, the above nucleotide sequence is used to obtain a plasmid vector with the sequence of SEQ ID No. 1, and the restriction site on the plasmid vector is BglII / ApaI.
[0010] Furthermore, in the application of the above nucleotide sequence, the sequences of the recombination identification primers are shown as SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6.
[0011] Furthermore, the application of the above nucleotide sequence includes the following steps: replacing the original promoter sequence in the specific nucleotide sequence primer vector plasmid, amplifying it through engineering bacteria transformation, and finally transfecting it into cells for expression.
[0012] Furthermore, the above nucleotide sequence is used to obtain a plasmid vector with the sequence of SEQ ID No. 1, and the restriction site on the plasmid vector is BglII / ApaI.
[0013] Furthermore, in the application of the above nucleotide sequence, the sequences of the recombination identification primers are shown as SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6.
[0014] Furthermore, the application of the above nucleotide sequence includes the following steps: knocking out the promoter sequence before the Luciferase gene in the original plasmid vector, amplifying it through transformation by engineering bacteria, and finally transfecting it into cells for expression.
[0015] Furthermore, the above promoter nucleotide sequence was knocked out, and the restriction site on the plasmid vector was BglII / ApaI.
[0016] Furthermore, the above promoter nucleotide sequence was knocked out, and the amplification and identification primer sequences used were shown in SEQ ID No. 7 and SEQ ID No. 8.
[0017] Furthermore, in the application of the above nucleotide sequence, the plasmid vector is PmirGLO.
[0018] Furthermore, in the application of the above nucleotide sequence, the engineered bacteria is DH5α. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 Plasmid map of PmirGLO vector.
[0020] Figure 2 The plasmid map of PmirGLO-Enhancer after cloning the nucleotide sequence into PmirGLO plasmid.
[0021] Figure 3 Relative fluorescence bar graph of the increased Luciferase mRNA expression level verified by RT-QPCR.
[0022] Figure 4 Relative fluorescence bar graph showing the increase in Luciferase protein expression level using dual luciferase validation.
[0023] Figure 5 The plasmid map of PmirGLO-Wt after the nucleotide sequence replaced the PmirGLO plasmid promoter.
[0024] Figure 6 The map of PmirGLO-Basic plasmid after the promoter of PmirGLO plasmid was cut.
[0025] Figure 7 Relative fluorescence bar graph showing the increase in Luciferase mRNA expression levels after reduction in RT-QPCR validation.
[0026] Figure 8 Relative fluorescence bar graph showing the increase in Luciferase protein expression level after reducing Luciferase protein expression using dual luciferase validation. DETAILED DESCRIPTION
[0027] The present invention is described in detail below with reference to examples. Unless otherwise specified, the experimental methods are all conventional methods, and the experimental materials used are all purchased from commercial channels.
[0028] The specific nucleotide sequence mentioned below is shown in SEQ ID No.1:
[0029]
[0030] 1. Cloning of nucleotide sequences, comprising the following steps:
[0031] 1.1 Construction of PmirGLO-Enhancer plasmid: After amplifying the nucleotide sequence (shown in SEQ ID No. 1), it was cloned into the upstream of the Luciferase gene promoter of the PmirGLO vector using the restriction site BglII / ApaI. The successfully connected vector was named PmirGLO-Enhancer vector. The constructed plasmid is shown in Figure 2 The nucleotide sequence of the primer pair Enhancer-F / Enhancer-R used for PCR amplification of the nucleotide sequence is as follows:
[0032] Enhancer-F: 5'-TCTGCTGCCGTGATCGCGCTGAACGCGTTTTAGCGGTGCGTACAATTAAGGGATTATGGTAAATCCACTTACTGTCTGCCCTCGTAGCCATCGA-3' (SEQ ID No. 2)
[0033] Enhancer-R: 5'-GAAAAGCGCCTCCCCTACCCGGTAGATCTCGATCCTCTACGCCGGACCATATGTGCAAGTATGTAAATATG-3' (SEQ ID No. 3)
[0034] The primer pair sequences for identifying the recombinant plasmid are as follows:
[0035] F: 5'-GATATCCAGATTGAAATCCT-3' (SEQ ID No. 4)
[0036] R1: 5'-CTTCACTCCAGCACACCCCT-3' (SEQ ID No. 5)
[0037] R2: 5'-CCCAGCGGGGCTGCTAAAGC-3' (SEQ ID No. 6)
[0038] 1.2 Construction of PmirGLO-Wt plasmid: After amplifying the nucleotide sequence (shown in SEQ ID No. 1), it was cloned into the upstream of the Luciferase gene promoter of the PmirGLO vector using the restriction site BglII / ApaI. The successfully connected vector was named PmirGLO-Wt vector. The constructed plasmid is shown in Figure 5 The nucleotide sequence of the primer pair Wt-F / Wt-R used for PCR amplification of the nucleotide sequence is as follows:
[0039] Wt-F: 5'-TCTGCTGCCGTGATCGCGCTGAACGCGTTTTAGCGGTGCGTACAATTAAGGGATTATGGTAAATCCACTTACTGTCTGCCCTCGTAGCCATCGA-3' (SEQ ID No. 2)
[0040] Wt-R: 5'-GAAAAGCGCCTCCCCTACCCGGTAGATCTCGATCCTCTACGCCGGACCATATGTGCAAGTATGTAAATATG-3' (SEQ ID No. 3)
[0041] The primer pair sequences for identifying the recombinant plasmid are as follows:
[0042] F: 5'-GATATCCAGATTGAAATCCT-3' (SEQ ID No. 4)
[0043] R1: 5'-CTTCACTCCAGCACACCCCT-3' (SEQ ID No. 5)
[0044] R2: 5'-CCCAGCGGGGCTGCTAAAGC-3' (SEQ ID No. 6)
[0045] 1.3 Construction of PmirGLO-Basic plasmid: The promoter before the Luciferase gene in the PmirGLO plasmid vector was removed using the restriction site BglII / ApaI and then reconnected. The successfully connected vector was named PmirGLO-Basic vector. Figure 6 The nucleotide sequence of the primer pair Basic-F / Basic-R used for PCR amplification and identification of the nucleotide sequence is as follows:
[0046] Basic-F: 5'-ACGATGCGTCCGGCGTAGAGGATCGAGATCTACCACCTGCAGCCCAAGCTTGGCAATCCGGTACTGTTGGTAAAG-3' (SEQ ID No. 7)
[0047] Basic-R: 5'-TCTTCGAGTGGGTAGAATGGCGCTGGGCCCTTCTTAATGTTTTTGGCATCTTCCATGGTGGCTTTACCAACAGTACCGGATTG-3' (SEQ ID No. 8)
[0048] The synthetic amplification of the specific nucleotide sequence and the ligation with the PmirGLO vector were completed by KeyGen Biotech Development Co., Ltd.
[0049] 2. Plasmid activity detection, detecting the effect of the inserted sequence by detecting the expression of the reporter gene, including the following steps:
[0050] 2.1 The PmirGLO plasmid and the constructed PmirGLO-Enhancer were transfected into 293 cells respectively. After 48 hours of transfection, the Dual Luciferase Reporter Assay Kit (Novagen) was used for detection according to its instructions.
[0051] The experimental results are as follows Figure 4 As shown, after inserting the nucleotide sequence, the PmirGLO-Enhance vector showed enhancer activity compared with Yangshen.
[0052] 2.2 After RNA was extracted from the remaining cell lysate in 2.1 by Trizol method, the mRNA expression level of Luciferase in cells was detected by RT-QPCR. Figure 3 As shown, after transfection with PmirGLO-Enhancer, the mRNA expression level of Luciferase in cells was higher than that in Yangshen.
[0053] 2.3 The constructed PmirGLO-Basic plasmid and PmirGLO-Wt were transfected into 293 cells respectively. After 48 hours of transfection, the Dual Luciferase Reporter Assay Kit (Novagen) was used for detection according to its instructions.
[0054] The experimental results are as follows Figure 8 As shown, after inserting a specific nucleotide sequence upstream of the original promoter, the Luciferase expression of the PmirGLO-Wt vector was lower than that of the vector without a promoter.
[0055] 2.4 After RNA was extracted from the remaining cell lysate in 2.3 by Trizol method, the mRNA expression level of Luciferase in cells was detected by RT-QPCR. Figure 8 As shown, after transfection with PmirGLO-Wt, the mRNA expression level of Luciferase in the cells was lower than that of PmirGLO-Wt.
Claims
1. A method for reducing background and increasing Luciferase gene expression activity, characterized in that: Introducing a nucleotide sequence before the Luciferase promoter can increase the background expression level of the Luciferase protein. When the Luciferase promoter is replaced with this sequence, the expression level of the Luciferase protein is lower than that of the control group without the promoter.
2. The specific nucleotide sequence according to claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID No.
1.
3. The primer pair for a specific nucleotide sequence according to claim 1, characterized in that: The nucleotide sequences of the primer pair are shown in SEQ ID No.2 and SEQ ID No.
3.
4. The specific nucleotide sequence according to claim 1, characterized in that: Using the synthesized DNA as a template, PCR amplification was performed using the primer pair described in SEQ ID No. 2 and SEQ ID No. 3 to obtain the nucleotide sequence shown in SEQ ID No.
1.
5. The use of the nucleotide sequence according to claims 1-4, characterized in that: The method comprises the following steps: a specific nucleotide sequence is introduced into a vector plasmid promoter, amplified by transformation through engineering bacteria, and finally transfected into cells for expression.
6. Use of the nucleotide sequence according to claim 5 to obtain a plasmid vector with SEQ ID No. 1 sequence, characterized in that: The restriction site is BglII / ApaI.
7. The use of the nucleotide sequence according to claim 5, characterized in that: The sequences of the recombination identification primers are shown in SEQ ID No.4, SEQ ID No.5 and SEQ ID No.
6.
8. The use of the nucleotide sequence according to claims 1-4, characterized in that: The method comprises the following steps: replacing the original promoter sequence in the vector plasmid with a specific nucleotide sequence primer, amplifying through transformation by engineering bacteria, and finally transfecting into cells for expression.
9. Use of the nucleotide sequence according to claim 8 to obtain a plasmid vector with SEQ ID No. 1 sequence, characterized in that: The restriction site is BglII / ApaI.
10. The use of the nucleotide sequence according to claim 8, characterized in that: The sequences of the recombination identification primers are shown in SEQ ID No.4, SEQ ID No.5 and SEQ ID No.
6.
11. The use of the nucleotide sequence according to claims 1-4, characterized in that: The method comprises the following steps: knocking out the promoter sequence before the Luciferase gene in the original plasmid vector, amplifying the gene through transformation by engineering bacteria, and finally transfecting the gene into cells for expression.
12. The use of the nucleotide sequence according to claim 11 to obtain a plasmid vector with the original promoter sequence knocked out, characterized in that: The restriction site is BglII / ApaI.
13. The use of the nucleotide sequence according to claim 11, characterized in that: The sequences of the amplification and identification primers used are shown in SEQ ID No.7 and SEQ ID No.
8.
14. Use of the nucleotide sequence according to claims 5, 8, and 11, characterized in that: The plasmid vector is PmirGLO.
15. Use of the nucleotide sequence according to claims 5, 8, and 11, characterized in that: The engineered bacteria is DH5α.