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Nattokinase and its coding gene and clone and expressing method

A technology of nattokinase and expression method, which is applied in the fields of botanical equipment and method, biochemical equipment and method, genetic engineering, etc., can solve the problem of low yield of active product, many purification steps, time-consuming and labor-intensive denaturation and renaturation, etc. problems, to achieve broad industrial application prospects, high fibrinolytic activity, and good stability.

Active Publication Date: 2006-08-09
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, nattokinase is mainly derived from the fermentation of wild Bacillus subtilis, the yield is low, the culture period is long, the medium composition is complicated, the purification steps are many, the yield is low, and the production cost is high
However, the existing nattokinase expressed in Escherichia coli is inactive inclusion body, denaturation and renaturation is time-consuming and labor-intensive, and the yield of active product is extremely low

Method used

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  • Nattokinase and its coding gene and clone and expressing method
  • Nattokinase and its coding gene and clone and expressing method
  • Nattokinase and its coding gene and clone and expressing method

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Embodiment 1, the separation and identification method of Bacillus subtilis YF38 CGMCC No.1308

[0037] 1. Isolation and identification of nattokinase-producing bacteria

[0038] 1. Isolation of nattokinase-producing bacteria

[0039] Select five kinds of fermented soybeans produced by Yunnan Yimen Food Factory, Guangdong Heshan Xingqiu Food Factory, Sichuan Yongchuan Rongmei Food Factory and Sichuan Fushun Shunding Food Factory Shunyi Branch, and separate natto from different products as follows Kinase-producing bacteria: Grind an appropriate amount of tempeh, dilute with sterile water, centrifuge, spread the supernatant on an LB plate, incubate at 37°C for 8-12 hours, pick a single colony, and streak it on the plate Store it, and at the same time inoculate it in a test tube containing LB liquid medium and culture it with shaking at 37°C for 8-12 hours. Finally, the culture supernatants of single colonies isolated from the five products were tested for fibrinolytic a...

Embodiment 2

[0046] Embodiment 2, the cloning of nattokinase whole gene

[0047] 1. PCR amplification of the whole gene of nattokinase

[0048] According to known nattokinase gene sequence design primer, primer sequence is as follows (primer sequence both ends comprise restriction endonuclease Eco RI and Sal I recognition site):

[0049] Primer 1: 5′-gcg gaa ttc gta tga aaa tag tta-3′

[0050] Primer 2: 5′-gta gtc gac tcc ggt gct tgt gaa-3′

[0051]Using the total DNA of the YF38 CGMCC No.1308 strain as a template, under the guidance of primers 1 and 2, the nattokinase gene was amplified by PCR. The PCR reaction conditions were: 94°C for 1min, 54°C for 1min, 72°C for 1.3min, A total of 30 cycles. After the reaction, 2 μL of the PCR product was taken for 1% agarose gel electrophoresis detection, and there was an obvious band at 1900 bp in ultraviolet detection, and its molecular weight was consistent with the size of the whole gene of nattokinase.

[0052] 2. Construction and identifica...

Embodiment 3

[0054] Embodiment 3, the expression of nattokinase of the present invention in escherichia coli

[0055] 1. Construction of Escherichia coli secretory expression vector pET26b-nkp

[0056] Design primers according to the coding sequence of the leading peptide of nattokinase and the mature peptide, the primer sequences are as follows: the two ends of the primer sequence contain restriction endonuclease EcoRI and SalI recognition sites):

[0057] Primer 3: 5′-gac gaa ttc gat ggc cgg aaa aag cag t-3′

[0058] Primer 4: 5′-gta gtc gac tcc ggt gct tgt gaa-3′

[0059] Using the vector pUC18-nkl containing the whole gene of nattokinase as a template, PCR amplification was carried out under the guidance of primers 3 and 4. The amplification conditions were: 94°C for 1 min, 54°C for 1 min, and 72°C for 1 min, a total of 30 samples cycle. After the reaction, the PCR product was precipitated and digested, and then the digested product was purified and then digested with the same enzym...

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Abstract

This invention discloses a natto kinase, its coding gene and its application, in which, said natto kinase is a protein including one of the sequences of the following amino acid residues, 1), SEQ ID No: 1in the list, 2), the amino acid residue sequence of the SEQ ID No:1 in the list is replaced, deleted and added by 1-10 amino acid residues and has fibrinolysis activity. The expression method is to set up a secretory expression carrier of colibacillus with the natto kinase gene to be introduced into a colibacillus to get a re-structured colibacillus to be induced to express the natto kinase gene, which has high fibrinolysis activity.

Description

technical field [0001] The invention relates to a protein and its encoding gene, cloning and expression method, in particular to a nattokinase, its encoding gene, its cloning and expression method. Background technique [0002] Nattokinase is a serine protease derived from natto, a traditional fermented food. Recent studies have shown that it has strong fibrinolytic activity and is a potential thrombolytic drug. It not only has a good thrombolytic effect, but also achieves the purpose of fibrinolysis through oral administration, and can promote the increase of endogenous t-PA at the same time. And it has the advantages of long drug effect time, no antigenicity, and can activate the organism's own fibrinolytic enzyme system. It can gently and continuously improve the fibrinolytic activity of blood, and achieve convenient, safe and effective treatment for cardiovascular and cerebrovascular embolism diseases. Prevention and treatment, its characteristics are better than curren...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/12C12N15/54C12N15/70
Inventor 钟瑾梁小波贾士芳孙玉芳陈美玲陈秀珠还连栋
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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