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Heparinase I

A technology of heparinase and recombinant vector, which is applied in the field of biological genetic engineering and fermentation engineering, can solve the problems of large loss of enzyme activity, complex process, high cost, etc., and achieve the effect of improving the stability of enzyme activity

Pending Publication Date: 2022-03-15
刘颖
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Natural heparanase I is usually purified from the fermentation broth of Flavobacterium heparinus, usually requires multi-step chromatographic purification, the enzyme activity is greatly lost, the yield is low, and it is difficult to exceed 10%, and its inducing additives At present, heparin sodium can only be extracted from the small intestinal mucosa of animals (mainly pigs and cattle). The process is complicated and the cost is high, which seriously limits the production and application of heparinase I.

Method used

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Examples

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preparation example Construction

[0049] The present invention provides a preparation method of heparanase I, the preparation method comprising the following steps: first synthesizing the nucleotide sequence encoding the heparanase I involved in this paper, and then recombining the nucleotide sequence with eukaryotic cells to express The vectors are combined to obtain a recombinant vector; the recombinant vector is transformed into a host cell, then induced to express, and then purified to obtain heparanase I.

[0050] In a preferred embodiment of the present invention, in the above preparation method, the steps of synthesizing the nucleotide sequence encoding the heparanase I involved in this paper are as follows:

[0051] First, reverse-translate the amino acid sequence of the original heparanase I into a nucleotide sequence. Preferably, the obtained nucleotide sequence should be preferred by the selected host cell. In the present invention, the translated nucleotide sequence is The nucleotide sequence obtai...

Embodiment 1

[0076] Example 1 Improves the nucleotide sequence optimization of heparanase I and constructs an expression vector

[0077] (a) The amino acid sequence of the original heparanase I is derived from the published sequence of heparanase I in the NCBI database. The URL of the NCBI database is: https: / / www.ncbi.nlm.nih.gov / protein / ACB38160.1 .

[0078] (b) Suzhou Jinweizhi Biotechnology Co., Ltd. reverse-translated the protein sequence into a DNA sequence according to the codon usage preference of Pichia pastoris in the Pichia pastoris codon preference data table, so that the codons of the DNA sequence Preferred by Pichia pastoris.

[0079] (c) Perform mutation modification on potential protease cleavage sites, and perform full sequence synthesis of the modified sequences respectively to obtain mutant nucleotide sequences.

[0080] (d) On the pPink-HC recombinant expression vector, two restriction sites, EcoRI and EcoRV, were selected to disconnect the expression vector, and the...

Embodiment 2

[0081] Example 2 Competent preparation and electrotransformation

[0082] Follow the operating instructions of the PichiaPink system kit.

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Abstract

The invention discloses heparinase I, a coding nucleotide sequence thereof, a recombinant vector comprising the nucleotide sequence, a host cell comprising the nucleotide sequence and application of the heparinase I. According to the heparinase I, site-directed mutagenesis is carried out on an amino acid sequence of existing heparinase I, and particularly, glutamine (Q) at the 42nd site, the 102nd site and the 209th site of the amino acid sequence of the existing heparinase I is subjected to site-directed mutagenesis into alanine (A). Compared with the existing heparinase I, the heparinase I obtained after mutation has better stability under the condition that the activity of the heparinase I is not influenced.

Description

technical field [0001] The invention widely relates to the fields of biological genetic engineering and fermentation engineering. In this field, the present invention relates to heparanase I and its coding gene, and the present invention further provides a method for preparing heparanase I using recombinant vectors and host cells. Background technique [0002] Heparinase is a kind of polysaccharide lyase that acts on heparin or heparansulfate. It is used to study the interaction between heparinase and its substrate polysaccharide heparin, which helps to elucidate the polysaccharide The mechanism of action of lyase, in analyzing the structure and biological function of complex mucopolysaccharides such as heparin, analyzing the mechanism of coagulation and anticoagulation in the human body, will only be used by low-molecular anticoagulant drugs and low-molecular anticoagulant drugs. Heparin has important uses in clinical and many other aspects. Heparanase is found in many mi...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/81C12N1/19C12R1/84
CPCC12N9/88C12N15/815C12Y402/02007
Inventor 刘颖
Owner 刘颖
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