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Plasmid for efficiently expressing porcine CRTC3, and construction method of adenovirus and application

A high-efficiency expression and construction method technology, applied in the direction of virus, virus/bacteriophage, double-stranded DNA virus, etc., can solve the problem of little research on the function of pig CRTC3 gene, achieve good tracer effect and simple construction method

Active Publication Date: 2020-03-06
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The current research focuses on the gene function of mouse CRTC3, but there is no report on the full-length gene of porcine CRTC3, and there are few studies on the gene function of porcine CRTC3

Method used

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  • Plasmid for efficiently expressing porcine CRTC3, and construction method of adenovirus and application
  • Plasmid for efficiently expressing porcine CRTC3, and construction method of adenovirus and application
  • Plasmid for efficiently expressing porcine CRTC3, and construction method of adenovirus and application

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Embodiment Construction

[0073] The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:

[0074] (1) Full-length cloning of porcine CRTC3 gene

[0075] 1. Design 3' RACE linker primers and specific downstream primers according to the predicted sequence on NCBI, respectively:

[0076] Adapter1: 5-cGAAAGCGACAAGGCCGTGATCCCGAAAGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3

[0077] Adapter2: 5-cGAAAGCGACAAGGCCGTGATCCCGAAAGC-3

[0078] CRTC3-1:5-GCCACAGGACTCCTTTCATTTG-3

[0079] CRTC3-2: 5-CACCAGCCTGACAGAAGACTCG-3

[0080] 2. According to the first strand cDNA synthesis kit (Thermo k1621), perform nested PCR reaction:

[0081] (1) The first round of PCR reaction system is as follows:

[0082] Reaction solution composition volume Taq plus DNA Polymerase (5U / μL) 1μL 10×Taq plus PCR Buffer 5μL dNTP Mixture (2.5mM each) 4μL Oligo(dT) 1μL CRTC3-1 (10pM) 2μL Adaptor...

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Abstract

The invention discloses a construction method of an efficient expression vector pAdM-FH-GFP-CRTC3 of a pig CRTC3. The construction method comprises the following steps: cloning a pig CRTC3 sequence; constructing an AdM-FH-GFP (adenovirus) vector; inserting the porcine CRTC3 gene into the multiple cloning sites of the pAdM-FH-GFP adenovirus vector; and packaging the pAdM-FH-GFP adenovirus so as toobtain the pAdM-FH-GFP adenovirus. The invention also discloses application of a porcine CRTC3 high-efficiency expression vector, which is used for researching and tracing the positioning of the pig CRTC 3 in different cells, and after the constructed pAdM-FH-GFP-CRTC 3 is transfected to 293T cells and subcutaneous fat cells of pigs, the CRTC 3 subcellular positioning is observed under a fluorescence microscope.

Description

technical field [0001] The invention relates to a pAdM-FH-GFP-CRTC3 plasmid, an adenovirus, a construction method and a use thereof for highly expressing porcine CRTC3 (cAMP responsive element bindingregulated transcription coactivators), which belong to the application technology in the field of biology and modern agricultural technology. Background technique [0002] Energy metabolism is a process from the intake of nutrients from the outside to the decomposition and utilization of cells in the body, in which cAMP participates in the regulation of cellular energy conversion and utilization, and plays an important role in the life activities of the body. Studies have found that the cAMP-PKA-mediated signaling pathway regulates the transcription and translation of downstream target genes through the joint action of the cAMP response element binding protein CREB and its co-activator CRTC. CREB-regulated transcriptional coactivator 3 (CRTC3) is a member of the CRTC family and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N15/65C12N7/01
CPCC12N7/00C12N15/65C12N15/86C12N2710/10021C12N2710/10043
Inventor 单体中刘嘉琪汪以真
Owner ZHEJIANG UNIV
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