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Expression plasmid system of recombinant Sushi polypeptide, and construction method and application thereof

A technology for expressing plasmids and construction methods, which is applied in the field of preparation of recombinant Sushi polypeptides, can solve the problems of restricting the mass production and application of recombinant proteins, and achieve the effects of large-scale production and application and low-cost expression

Inactive Publication Date: 2013-12-11
SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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Problems solved by technology

Although the expression of recombinant proteins in Escherichia coli has the advantages of rapidity, large quantities and low cost, the large-scale production and application of such recombinant proteins are severely limited due to the presence of LPS in Escherichia coli

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  • Expression plasmid system of recombinant Sushi polypeptide, and construction method and application thereof
  • Expression plasmid system of recombinant Sushi polypeptide, and construction method and application thereof
  • Expression plasmid system of recombinant Sushi polypeptide, and construction method and application thereof

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Embodiment Construction

[0035] Generally speaking, as one aspect of the present invention, the protein trans-cleavage system is mainly used to express the CES12 region of Limulus factor C, and the protein obtained by the cleavage has endotoxin binding activity, which can be realized in the Escherichia coli system at low cost. Develop endotoxin detection reagents.

[0036] Further speaking, the present invention utilizes the protein trans-shearing system to express the CES12 gene of Limulus factor C. During the construction process of the protein trans-shearing system, the selected break site can be in Sushi1 of Limulus factor C and The 34 amino acid region for endotoxin binding, and the two precursor proteins expressed in segments using this protein trans-cleavage system do not have LPS binding activity, while the target protein CES12 obtained after cleavage has endotoxin neutralizing activity .

[0037]As a more preferred embodiment of the present invention, the establishment process of the protein...

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Abstract

The invention discloses an expression plasmid system of recombinant Sushi polypeptide, and a construction method and an application thereof. A typical embodiment of the invention may comprise the steps of disconnecting functional segments capable of binding with endotoxin in limulus factor C at specific sites by using a protein in-vitro shearing system; connecting the functional segments to N-terminal segments (IN) and C-terminal segments (IC) of split intein respectively for expression in escherichia coli; and in-vitro inducing the intein to shear after an expressed product is subjected to purification, renaturation and endotoxin removal, so as to obtain CES12 segments in C factor, the CES12 segments being recombinant Sushi polypeptide with an endotoxin-binding activity. The expression plasmid system can realize rapid, large and low-cost expression of the functional segments with the endotoxin-binding activity in the limulus factor C, can effectively prevent influences of LPS contained in host cells, and is beneficial to large-scale production and application of low-cost endotoxin-detecting reagents.

Description

technical field [0001] The invention relates to a recombinant polypeptide, in particular to a preparation method of a recombinant Sushi polypeptide. Background technique [0002] Bacterial endotoxin is a unique structure on the outer layer of the cell wall of Gram-negative bacteria, and its chemical composition is lipopolysaccharide (LPS). Bacterial endotoxin can cause fever, shock and even death in the human body, but it is ubiquitous and not easy to inactivate, so endotoxin detection is particularly important in pharmaceutical and medical device manufacturing. In addition, endotoxin detection can also be used in the diagnosis and prevention of clinically relevant diseases. [0003] Turtle reagent is the most commonly used reagent for detecting endotoxin in the world so far. However, due to limitations such as the limited number of Limulus and the inhomogeneity of Limulus reagent batches, it is urgent to develop a substitute for Limulus reagent. Studies have shown that Li...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/66C12N9/64C12N1/21
Inventor 张春王影影齐兴梅刘涛祁静
Owner SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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