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Prokaryotic expression vector for recombinant protein of casein kinase I(alpha) and construction method thereof

A casein kinase and prokaryotic expression technology, applied in the field of prokaryotic expression vector construction, can solve the problem of casein kinase Iα not having a relatively mature solution, and achieve the effect of easy implementation, mature system and complete structural domain

Inactive Publication Date: 2018-06-05
ZHUJIANG HOSPITAL SOUTHERN MEDICAL UNIV
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

[0004] How to use the prokaryotic expression system to obtain more and more stable casein kinase Iα from eukaryotes has not yet been matured

Method used

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  • Prokaryotic expression vector for recombinant protein of casein kinase I(alpha) and construction method thereof
  • Prokaryotic expression vector for recombinant protein of casein kinase I(alpha) and construction method thereof
  • Prokaryotic expression vector for recombinant protein of casein kinase I(alpha) and construction method thereof

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Embodiment Construction

[0038] The present invention will be described in further detail below in conjunction with the accompanying drawings and specific embodiments.

[0039] The construction method of the prokaryotic expression vector of the recombinant protein of casein kinase Iα of the present invention, carries out following steps successively:

[0040] (1) Primer design

[0041] According to the principle of primer design, search the GenBank resource library of the National Library of Medicine online, design PCR primers according to the CKIα sequence (NM_053615) (SEQ ID NO: 1) on GenBank, amplify the target gene CKIα, and add NdeI, a BamHI site was added to the 3' end.

[0042] Upstream primer 5'-GGGAATTCCATATGATGGCGAGCTCCAGCGGTA-3' (SEQ ID NO: 3)

[0043] Downstream primer 5'-CGCGGATCCAAAGCCCGTCGGGGTCTG-3' (SEQ ID NO: 4)

[0044] (2) Acquisition of rat hepatocyte cDNA

[0045] Put the fresh rat liver tissue into a mortar, add an appropriate amount of liquid nitrogen, and grind it; add 1.0m...

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Abstract

The invention discloses a prokaryotic expression vector for a recombinant protein of casein kinase I(alpha). The prokaryotic expression vector contains a nucleotide sequence as shown in SEQ ID No. 1,a restriction enzyme cutting site Nde I and a restriction enzyme cutting site BamH I. A construction method for the prokaryotic expression vector for the recombinant protein of the casein kinase I(alpha) comprises the following steps: designing primers used for amplifying CKI(alpha) according to the nucleotide sequences of mouse casein kinase I(alpha) included in the GenBank; acquiring the whole cDNAs of mouse cells; cloning a CKI(alpha) gene in the cDNAs by using the primers used for amplifying CKI(alpha); and connecting the cloned CKI(alpha) gene with a prokaryotic expression vector so as toobtain the recombinant prokaryotic expression vector. The prokaryotic expression vector for the recombinant protein of the casein kinase I(alpha) and the construction method thereof successively utilize a prokaryotic expression system to prepare recombinant CKI(alpha).

Description

technical field [0001] The invention relates to a prokaryotic expression vector construction technology, in particular to a prokaryotic expression vector of a recombinant protein of casein kinase Iα and a construction method thereof. Background technique [0002] Casein kinase Iα is a member of the CKI family. It is a serine / threonine protein kinase with unique physical and chemical properties. It exists widely in eukaryotic organisms from yeast to humans, and is mainly distributed in the cytoplasm, cell membrane, nucleus and cytoskeleton. sites, and during mitosis, also localizes to synaptic vesicles, centrosomes, and spindle microtubules. Constitutively active (ie, both isolated and prokaryotically expressed CKIα are active). CKIα can phosphorylate the phosphorylated site in the substrate protein, and can also be phosphorylated as the substrate of other protein kinases, participate in the cascade phosphorylation process of the protein, play a role in the signal transducti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/66C12N9/12
CPCC12N9/12C12N15/66C12N15/70C12N2800/101C12Y207/11001
Inventor 蔡磊高毅潘明新李阳
Owner ZHUJIANG HOSPITAL SOUTHERN MEDICAL UNIV
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