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Double-fluorescence report recombinant plasmid vector as well as construction method and application thereof
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A recombinant plasmid, dual-fluorescence technology, applied in the application field of the 3' untranslated region, can solve the problems of limited use range, expensive luciferase substrate, test processing requirements, differences in test characteristics, etc., to achieve the effect of avoiding errors.
Inactive Publication Date: 2018-02-13
SHAANXI NORMAL UNIV
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However, traditional co-reporter genes such as chloramphenicolacetyltransferase (CAT), β-galactosidase (β-Gal) or glucuronidase (GUS) are not convenient because of their respective test processing requirements and detection characteristics. difference
In recent years, firefly luciferase has been widely used to avoid the above disadvantages, but due to the high price of luciferase substrates, the need to use special instruments to detect luciferase expression and other factors limit its scope of use.
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Embodiment 1
[0046] Embodiment 1: Construction of recombinant plasmid vector
[0053] HEK293 cells, a human embryonic kidneycell line, were purchased from the Cell Center of the Institute of Basic Medical Sciences, Peking Union Medical College, China.
[0054] 1.2 Reagents
[0055] The restriction endonucleases used, T4DNA ligase were purchased from Thermo Fisher Scientific, USA. Primerstar polymerase was purchased from Clontech, USA;
[0056] Plasmid small / medium extraction and purification kits, DNA purification kits, DNA...
Embodiment 2
[0097] Example 2: Cell-level experiment of the recombinant plasmid vector pTRE3G-BI-GFP-mCherry-3UTR to verify the relationship between miR-23b and its target gene
[0101] Human embryonic kidney 293 cells: ATCC, USA.
[0102] 2.3 Experimental materials and reagents
[0103] RPMI 1640 culture medium and fetal bovine serum were purchased from Gibco Company of the United States, Lipofectamine 2000 was purchased from Lifetechnologies Company of the United States, and cell culture dishes were purchased from Fisher Scientific Company of the United States.
[0104] 2.4 Experimental plasmid
[0105] pTRE3G-BI-GFP-mCherry-3UTR was constructed according to Example 1.
[0106] pCMV-Tet3G was purchased from Clontech Laboratories, USA.
[0107] miR-23b mimic and control miRNA mimic were purchased from Ribobio.
[0108] 2.5 Experimental method
[0109] The day ...
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Abstract
The invention discloses a double-fluorescence report recombinant plasmid vector. The double-fluorescence report recombinant plasmid vector contains a coded green fluorescenceproteingene sequence, ared fluorescence proteingene sequence and a 3' non-translation area sequence of a miRNA specific target gene. The construction method comprises the following steps: amplifying a green fluorescence proteingene and a red fluorescence protein gene by a PCR method; inserting the amplified green fluorescence protein gene segment into the 3' direction of a plasmid vector pTRE3G-BI promoter to obtain arecombinant plasmid vector pTRE3G-GFP, and performing sequencing analysis; inserting the amplified red fluorescence protein gene segment into the 5' direction of the plasmid vector pTRE3G-BI promoterto obtain a recombinant plasmid vector pTRE3G-GFP-mCherry, and performing sequencing analysis; amplifying the 3' non-translation area sequence of the target gene by the PCR method and inserting in the mCherry 3'direciton of the plasmid vector pTRE3G-GFP-mCherry; naming the recombinant plasmid vector which is verified to be correct as pTRE3G-GFP-mCherry-3UTR. The double-fluorescence report recombinant plasmid vector can be applied to verify whether miRNA and the target gene thereof have mutual combination relation in the sequence or not.
Description
technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a dual-fluorescent reporter recombinant plasmid vector and a construction method thereof, and an application for verifying whether miRNA binds to the 3' untranslated region of its specific target gene. Background technique [0002] MicroRNA (miRNA) is a class of non-coding single-stranded RNA molecules encoded by endogenous genes with a length of about 22 nucleotides. They participate in post-transcriptional gene expression regulation in animals and plants. Each miRNA can have multiple target genes, and several miRNAs can also regulate the same gene. This complex regulatory network can not only regulate the expression of multiple genes through one miRNA, but also finely regulate the expression of a gene through the combination of several miRNAs. It is assumed that miRNAs regulate one-third of human genes. From the beginning of this century, scientists be...
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