Human STING gene promoter inhibition regulatory region and application thereof
A promoter and gene technology, which is applied in the field of repression and regulatory elements in the promoter region of the human STING gene, can solve problems such as the research report on the repression and regulation region of the promoter of the STING gene that has not yet been seen.
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Embodiment 1
[0034] Example 1 Construction of the STING 5' end promoter activity suppression region recombinant plasmid and its deletion mutant Determination of the key region for inhibiting the STING 5' end promoter activity
[0035] 1. Extract genomic DNA when HEK293T cells in logarithmic growth phase grow to 90% full.
[0036] 2. Design a pair of primers using Oligo 7 software. KpnI and BglII restriction sites were loaded into the upstream and downstream primers, respectively. The primer sequences are as follows:
[0037] Table 1
[0038]
[0039] 3. Using the whole genome DNA of HEK293T cells as a template, adding the above primers, using EXtaq (Takara, Japan) as a polymerase to configure a PCR system, and cloning fragments of the STING gene promoter repression region with different lengths. The conditions are as follows: pre-denaturation at 95°C for 5min, denaturation at 95°C for 30s, annealing at 58-62°C for 30s, extension at 72°C for 2min. The three steps of denaturation, ann...
Embodiment 2
[0047] Example 2 The fragment of the human STING promoter activity inhibitory sequence was subcloned forward and reverse into the pGL3-Control vector, and its effect on inhibiting transcriptional activity was detected
[0048] 1. Use Oligo 7 software to design a pair of primers. The HindⅢ restriction site is loaded into the upstream or downstream primers respectively.
[0049] The primer sequences are as follows:
[0050] Table 2
[0051]
[0052] 2. Use the DNA of the STING 5-1a plasmid as a template, add the above primers, use EXtaq (Takara, Japan) as a polymerase to configure a PCR system, and clone the forward and reverse fragments of the STING gene promoter repression region. The obtained PCR product was subjected to agarose gel electrophoresis, and the electrophoresis band was similar in size to the target fragment, and the PCR product was purified and recovered by gel cutting. PCR product and pGL3-Basic plasmid ( Image 6 ) to carry out a single enzyme digestion ...
Embodiment 3
[0054] 4. Results such as Image 6 It shows that the inhibitory fragment of +169~+267 in the 5' upstream of the human STING gene in pGL3-Control STING 5-1b, whether it is a forward sequence vector or a reverse sequence vector transfected into HEK293T and HEPG2 cells, can lead to pGL3- The decrease of the promoter activity of the Control plasmid was more than 10 times, and the difference was statistically significant. Example 3 Forward and reverse subcloning of the repressed core region sequence into the cGAS promoter plasmid vector to detect transcriptional activity
[0055] As described in Example 2, the human STING repression regulatory region was also subcloned forward and reverse to the cGAS promoter plasmid ( Figure 7 Schematic diagram, the previous research group has proved that the plasmid has promoter activity). Then a dual luciferase activity assay was performed. Such as Figure 8The results showed that compared with the control group pGL3-Basic, when the inhibit...
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