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Eukaryotic expression method of sea cucumber cathepsin

A cathepsin and expression method technology, applied in the field of eukaryotic expression of sea cucumber cathepsin, can solve problems such as gene screening of sea cucumber cathepsin, functional research has not been systematically carried out, etc. Effects of important economic and social values

Inactive Publication Date: 2016-03-09
ZHEJIANG OCEAN UNIV
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Problems solved by technology

However, the screening and functional research of sea cucumber cathepsin genes and the development of related protease products have not been carried out systematically.

Method used

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  • Eukaryotic expression method of sea cucumber cathepsin

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Embodiment 1

[0036] A method for eukaryotic expression of sea cucumber cathepsin, comprising the steps of cloning the nucleotide sequence of sea cucumber cathepsin into a eukaryotic expression vector, transforming host cells, and inducing and expressing, the specific steps are as follows:

[0037] (1) Full-length cloning of sea cucumber cathepsin gene

[0038] According to the obtained EST sequence fragment of A. japonicus cathepsin gene, specific full-length cloning primers were designed, and the gene fragment was amplified by RACE technology to obtain the full-length cDNA sequence of the functional gene. The cDNA sequence was initially analyzed by NCBI website and protein translation software. correctness.

[0039] (2) Prediction of protein structure analysis and determination of gene fragments expressed in eukaryotes

[0040] Find and determine the open reading frame of the full-length sequence of the sea cucumber cathepsin gene, determine the coding sequence and deduce the amino acid ...

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Abstract

The invention relates to an eukaryotic expression method of sea cucumber cathepsin. The method comprises the following steps: screening a cathepsin gene EST sequence on the basis of an Apostichopus japonicus transcriptome database obtained through early stage working according to a bioinformatics analysis result; acquiring gene full-length sequence information by adopting an RACE full-length cloning technology on the basis of a fragment sequence; predicating and analyzing the structure characters and function activity of an encoded protein according to the full-length sequence information, and searching and determining a key structural domain; determining a gene sequence fragrance accessed to an expression system according to the predication and analysis result; constructing a Pichia pastoris eukaryotic expression system, and expressing a target protein in vitro; and detecting and explicating the activity and function characters of the protease obtained after expression. The sea cucumber protease expressed through the method has the characteristics of low production cost, easy quality control, increase of the selectable kinds of collagen proteolysis proteases, increase of the enzymatic hydrolysis production benefit, and important economic and social values.

Description

technical field [0001] The invention relates to the field of biological protease preparation, in particular to a eukaryotic expression method of sea cucumber cathepsin. Background technique [0002] Procollagen is a structural protein of body tissue, and its many physiological functions have been systematically studied and widely recognized. With the rapid development of the collagen industry, the demand for protease, the key production factor in the collagen enzymatic hydrolysis process, is also increasing. At present, the commonly used enzymes generally have the disadvantage of low enzymatic hydrolysis efficiency of collagen, and the use of genetic engineering technology to develop new collagenase can make up for this fatal defect, and the quality of collagenase products produced by bioengineering technology is easy to control and the production cost is low. low. Apostichopus japonicus has a very strong autolysis ability, the essence of which is the efficient enzymolysis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N9/64C12Q1/68C12R1/84
Inventor 王天明李丹凤李明月王觅吕小康计振伟吴启本
Owner ZHEJIANG OCEAN UNIV
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