Construction and application of bovine CART gene eukaryotic overexpression vector

A technology for overexpressing vectors and genes, applied in the direction of nucleic acid vectors, vectors, and the use of vectors to introduce foreign genetic material, etc., can solve problems such as the infeasibility of regulatory mechanisms, and achieve the effect of structural integrity

Pending Publication Date: 2022-06-07
SHANXI AGRI UNIV
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Problems solved by technology

Commonly used expression vectors only construct the CDS region, and there is a problem that it is not feasible to study the regulation mechanism of miRNA on the mRNA 3′UTR

Method used

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  • Construction and application of bovine CART gene eukaryotic overexpression vector
  • Construction and application of bovine CART gene eukaryotic overexpression vector
  • Construction and application of bovine CART gene eukaryotic overexpression vector

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Embodiment Construction

[0075] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0076] see Figure 1-15 , the invention provides a technical solution: the construction and application of a bovine CART gene eukaryotic overexpression vector, which specifically includes the following steps:

[0077] Step 1: Use PCR method to obtain bovine CARTmRNA+UTR sequence fragment:

[0078] ①The upstream and downstream primers of the target gene are respectively added with the homologous sequences on both sides of EcoRI and B...

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Abstract

The invention discloses construction and application of a cattle CART gene eukaryotic overexpression vector. The construction specifically comprises the following steps: step 1, obtaining a cattle CART mRNA + UTR sequence fragment by using a PCR method; step 2, cloning a target gene CART mRNA + UTR into a vector pEX-3; step 3, overexpression of CART mRNA + UTR in the 293T cell: culturing the 293T cell; and the pEX-3-CART mRNA + UTR recombinant overexpression vector is used for transfecting a 293T cell. The method has the technical effects that the regulation of miRNA in primary cells on an mRNA UTR (Untranslated Region) region can be better simulated; a foundation is laid for researching a CART posttranscriptional regulation mechanism and a CART regulation and control bovine follicle development mechanism, and the overexpression condition of pEX-3-CART mRNA + UTR is detected through expression of CART mRNA and CART protein.

Description

technical field [0001] The invention relates to the technical field of gene expression vector construction and application, in particular to the construction and application of a bovine CART gene eukaryotic overexpression vector. Background technique [0002] Cocaine-and Amphetamine-Regulated Transcript Peptide (CART) is an endogenous neuropeptide widely distributed in animals and is involved in various biological functions such as feeding regulation, hormone release, drug dependence and follicular development. . Usually, the overexpression vector only constructs the CDS region of the gene on the vector, and the mRNA after cell transcription does not have a UTR region, so there is a problem that the regulation of mRNA UTR by miRNA cannot be studied. [0003] miRNA is a kind of non-coding single-stranded RNA with a length of about 22nt encoded by endogenous genes. In animals, in most cases, miRNA is not fully complementary to the 3'UTR of mRNA, thereby hindering the translat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/66C07K14/47
CPCC12N15/85C12N15/66C07K14/4702C12N2800/107C12N2840/105
Inventor 李鹏飞成俊丽杜海燕
Owner SHANXI AGRI UNIV
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