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Recombinant bacterium for producing sulfo-recombinant hirudin as well as preparation method and application of recombinant bacterium

A technology of recombinant hirudin and recombinant bacteria, applied in the field of genetic engineering, can solve the problems of reduced activity, increased pressure of downstream purification, increased production cost and difficulty, etc., to achieve the effect of improving activity and reducing the pressure of subsequent purification

Active Publication Date: 2021-11-26
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the lack of functional sulfonylase, most bioengineering hosts cannot complete the post-translational modification reaction of hirudin, which makes the activity of recombinant hirudin lacking sulfolation significantly lower than that of natural hirudin
Some studies can achieve sulfoylation of products through in vitro enzymatic reactions, but this greatly increases the production cost and difficulty, increases the pressure of downstream purification, and is difficult to achieve large-scale production

Method used

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  • Recombinant bacterium for producing sulfo-recombinant hirudin as well as preparation method and application of recombinant bacterium
  • Recombinant bacterium for producing sulfo-recombinant hirudin as well as preparation method and application of recombinant bacterium
  • Recombinant bacterium for producing sulfo-recombinant hirudin as well as preparation method and application of recombinant bacterium

Examples

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Effect test

Embodiment 1

[0028] Example 1. Preparation of recombinant bacteria for producing sulfo-recombinant hirudin.

[0029] The recombinant bacterium prepared in this embodiment overexpresses the sulfonylase gene and the hirudin gene, the nucleotide sequence of the hirudin gene is as shown in SEQ ID No.1, and the sulfonylase gene, the nucleoside The acid sequence is shown in SEQ ID No.4, and the starting strain is Escherichia coli. The build method is as follows:

[0030] 1) Hirudin gene expression vector construction: adding a nucleotide sequence encoding a periplasmic secreted peptide to the 5' end of the hirudin gene to obtain a hirudin gene with a periplasmic secreted peptide, and then connecting it to a plasmid vector, Obtain the expression vector expressing hirudin; The specific method is as follows:

[0031] The nucleotide sequence encoding the periplasmic secretory peptide shown in SEQ ID No.5 was added to the 5' end of the hirudin gene shown in SEQ ID No.1, and the obtained sequence wa...

Embodiment 2

[0041] Embodiment 2. repeat embodiment 1, and the difference with embodiment 1 is, the hirudin gene described in the present embodiment, nucleotide sequence is as shown in SEQ ID No.2, compared with embodiment 1, present embodiment In the hirudin gene, the 181st base G was replaced with the base T, and the 183rd base A was replaced with the base C. Refer to Example 1 for the remaining steps.

Embodiment 3

[0042] Embodiment 3. Repeat Example 1, the difference from Example 1 is that the hirudin gene described in this example, the nucleotide sequence is as shown in SEQ ID No.3, compared with Example 1, the present example In the hirudin gene, the 181st base G is replaced with a base T, the 183rd base A is replaced with a base C, the 184th base G is replaced with a base T, and the 186th base A is replaced with a base c. Refer to Example 1 for the remaining steps.

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Abstract

The invention discloses a recombinant bacterium for producing sulfo-recombinant hirudin as well as a preparation method and application of the recombinant bacterium, and belongs to the technical field of genetic engineering. In order to improve the synthesis yield of the sulfated recombinant hirudin and reduce the production cost, the invention provides the recombinant bacterium for producing the sulfo-recombinant hirudin, wherein the recombinant bacterium overexpresses a sulfotransferase gene and a hirudin gene, and an original strain is escherichia coli; and the nucleotide sequence of the hirudin gene is one of SEQ ID No.1 to SEQ ID No.3, and the nucleotide sequence of the sulfotransferase gene is as shown in SEQ ID No.4. According to the method, the activity of the recombinant hirudin is remarkably improved, a one-step purification method of the hirudin is realized, a recombinant hirudin product with relatively high purity can be obtained at one time, and the pressure of subsequent purification is greatly reduced.

Description

technical field [0001] The invention specifically relates to a recombinant bacterium for producing sulfo-recombined hirudin and its preparation method and application, belonging to the technical field of genetic engineering. Background technique [0002] Hirudin, secreted by the salivary glands of leeches, is one of the most potent natural thrombin inhibitors known. As a direct inhibitor of thrombin, hirudin is quite different from heparin, which is currently used clinically, in the principle of thrombin inhibition. Heparin inhibits the activity of thrombin by catalyzing other anticoagulants (such as antithrombin III or heparin cofactor II). It is an indirect anticoagulant drug that cannot exert its anticoagulant activity alone. Hirudin directly binds to the active site of thrombin through its special structure, thereby irreversibly losing its enzymatic activity. Compared with heparin, hirudin has higher anticoagulant activity, stronger specificity and stability, and will ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/15C12N15/54C12P21/02C12R1/19
CPCC07K14/815C12N9/13C12N15/70C12P21/02C12Y208/02C07K2319/036
Inventor 孙粤包子娴年锐莱东咸漠刘文帅樊喜英陈泉
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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