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Method for constructing and packaging bovine PDHB gene overexpression recombinant adenovirus vector

A recombinant adenovirus, gene overexpression technology, applied in the direction of virus/phage, double-stranded DNA virus, virus, etc., can solve the problems of difficult transfection and short expression time.

Inactive Publication Date: 2017-10-10
NORTHWEST A & F UNIV
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Problems solved by technology

[0003] In summary, the problems existing in the prior art are: the construction of transient expression vectors is easy, but there are problems such as difficult transfection in primary cells and short expression time

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  • Method for constructing and packaging bovine PDHB gene overexpression recombinant adenovirus vector
  • Method for constructing and packaging bovine PDHB gene overexpression recombinant adenovirus vector
  • Method for constructing and packaging bovine PDHB gene overexpression recombinant adenovirus vector

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Embodiment Construction

[0031] In order to make the object, technical solution and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

[0032] The application principle of the present invention will be described in detail below in conjunction with the accompanying drawings.

[0033] The sequence of Qinchuan bovine PDHB gene recombinant adenovirus vector provided in the embodiment of the present invention is: GenBank: AY370909.2 (https: / / www.ncbi.nlm.nih.gov / nuccore / AY370909.2).

[0034] Such as figure 1 As shown, the bovine PDHB gene overexpression recombinant adenoviral vector construction method provided by the embodiments of the present invention includes the following steps:

[0035] S101: Design primers according to the bovine PDHB gene mRNA sequence...

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Abstract

The invention belongs to the technical field of recombinant adenovirus vectors and discloses a method for constructing and packaging a bovine PDHB gene overexpression recombinant adenovirus vector. The sequence of the bovine PDHB gene overexpression recombinant adenovirus vector is GenBank: AY370909.2. Bovine PDHB gene CDS obtained through cloning is accordant with a sequence included in a database GenBank, the bovine PDHB gene CDS and a shuttle vector pAdTrack-CMV are recombined and are transfected with an HEK 293A cell, a recombinant adenovirus Ad-PDHB is successfully combined, and the titer of the recombinant adenovirus Ad-PDHB is 1.66*10<9>PFU.mL<1>; and after precursor fat cells in bovine muscles are infected by the adenovirus Ad-PDHB, the expression level of PDHB in mRNA is 25.5 times higher than that of a control group.

Description

technical field [0001] The invention belongs to the technical field of recombinant adenovirus vectors, in particular to a method for constructing bovine PDHB gene overexpression recombinant adenovirus vectors. Background technique [0002] Bovine pyruvate dehydrogenase β subunit (Pyruvate dehydrogenase β subunit, PDHB) gene catalyzes pyruvate into acetyl-CoA (acetyl-CoA), which is the key enzyme connecting glycolysis and TCA cycle metabolism. Studies have shown that the expression level of PDHB gene is positively correlated with the content of intramuscular fat (IMF). For gene function research, overexpression technology is mainly used. At present, there are two main types of vectors for introducing foreign genes into eukaryotic cells for expression. One is the non-viral expression vector system, the common one is pCDNA3.1(±) non-fusion eukaryotic cell expression vector, which has the advantages of high safety, low toxicity, unlimited length of foreign gene, and the disadv...

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Application Information

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IPC IPC(8): C12N15/66C12N15/861
CPCC12N15/66C12N15/86C12N2710/10343
Inventor 李安宁昝林森武丽娟王晓宇
Owner NORTHWEST A & F UNIV
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