Nattokinase expression method and its special expression vector and engineering bacterium

A technology of nattokinase and carrier, applied in the field of expression of nattokinase and its special expression vector and engineering bacteria, can solve the problems of high production cost, limitation of development and application of nattokinase, low yield of nattokinase, etc.

Inactive Publication Date: 2007-01-24
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 natto and fermented soya bean fermented by wild Bacillus subtilis, but the flavor of natto and fermented soya bean cannot be used and accepted by many people, and the yield of nattokinase isolated and purified from the fermentation broth is usually low , high production costs, these factors have limited the development and application of nattokinase to a certain extent

Method used

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  • Nattokinase expression method and its special expression vector and engineering bacterium
  • Nattokinase expression method and its special expression vector and engineering bacterium
  • Nattokinase expression method and its special expression vector and engineering bacterium

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1, the construction of special expression vector for nattokinase

[0031] 1. Construction of nattokinase constitutive expression vector

[0032] see figure 1 Construct the constitutive expression vector of nattokinase, the specific process comprises the following steps:

[0033] 1. Construction of intermediate vector pXB100 containing lactic acid bacteria promoter P32

[0034] Design primers according to the sequence of lactic acid bacteria promoter P32 (GenBank number: M24764), and add recognition sites for restriction endonucleases EcoRI and SacI at both ends of the primer sequences, and introduce three consecutive stop codons in the downstream primers sub and a lactic acid bacteria ribosome binding site to form a transcriptional fusion, the primer sequence is as follows: primer 1 (upstream primer): 5'-atc gaattc ggtcctcgggatatg-3' (the underlined base is the restriction endonuclease EcoRI recognition site)

[0035] Primer 2 (downstream primer): 5'-ac ...

Embodiment 2

[0063] Embodiment 2, the expression of nattokinase in lactic acid bacteria and the identification of expression product

[0064] 1. Expression of nattokinase in lactic acid bacteria

[0065] 1. Constitutive expression of nattokinase in lactic acid bacteria

[0066] The lactic acid bacteria constitutive expression vector pXB520 of nattokinase constructed in Example 1 was transformed into Lactococcus lactis L.lactis MG1363, and the GM17 resistance plate containing 5 μg / mL erythromycin (tryptone 0.5%, soybean peptone 0.5%, beef extract 0.5%, yeast extract 0.25%, glucose 0.5%, vitamin C 0.05%, β-sodium glycerophosphate 1.9%, magnesium sulfate 1mmol / L) screen positive transformants, extract the recombinant plasmid in the positive transformants, first The plasmid was identified by restriction endonuclease EcoRI, and then the plasmid was identified by restriction endonuclease KpnI+SacI, and the fragments of 1200bp and 3500bp were obtained by KpnI+SacI double digestion, which was in ...

Embodiment 3

[0075] Example 3, detection of fibrinolytic activity of nattokinase induced expression

[0076] Detect the fibrinolytic activity of the nattokinase induced expression of embodiment 2, the specific method is as follows:

[0077] First make a fibrin detection plate: take 10mL barbital sodium buffer (containing 0.05M barbital sodium, 0.09M NaCl, 0.0017M CaCl 2 , 0.0007M MgCl 2 ) to melt agarose (0.6%), and then take 10 mL of the same buffer solution to dissolve human fibrinogen (purchased from China Institute for Drug Evaluation and Testing) (20 mg / mL), and after incubating at 45 ° C for 45 min and 10 min, add 10 μ L of thrombin ( The solution (0.1BP / μL) (purchased from China National Institute for Drug Inspection and Identification) was mixed evenly, mixed with the above 20mg / mL human fibrinogen solution, poured into a horizontal sterile petri dish, left at room temperature for 1 hour, and punched with a punch . Take 30 μL sample and inject it into the small hole, incubate at...

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Abstract

The present invention discloses the expression method of natto kinase and its special expression vector and engineering bacterium. The expression vector is lactic acid bacteria constitutive expression vector containing constitutive expression promoter, natto kinase leading peptide and its mature peptide coding sequence; or lactic acid bacteria inducing expression vector containing inducing expression promoter, transmembrane signaling peptide coding sequence, natto kinase leading peptide and its mature peptide coding sequence. Experiment shows that the expressed product of the engineering bacterium has relatively high fibrinolysis activity up to 41.7 urokinase unit each milliliter of the fermented supernatant, eating safety, no toxic side effect on human body and high stability, is suitable for direct oral taking and may be added to various kinds of food. The present invention lays foundation for developing orally taken thrombolytic functional health food of natto kinase.

Description

technical field [0001] The invention relates to an expression method of nattokinase, a special expression vector and an engineering bacterium. Background technique [0002] Nattokinase (Nattokinase, NK, GenBank No.: AA065246) is a serine protease derived from natto, a traditional fermented food. Studies have shown that nattokinase has strong fibrinolytic activity and is a potential thrombolytic drug. It not only has a good thrombolytic effect, but also can achieve the purpose of fibrinolysis through oral administration, and can also promote endogenous tissue fibrinolysis. Lysosinogen activator (t-PA) increased. As a thrombolytic drug, nattokinase has the advantages of long duration of drug effect, no antigenicity, and activation of the organism's own plasminase system. It can gently and continuously improve the fibrinolytic activity of the blood, and achieves the goal of treating cardiovascular and cerebrovascular embolism diseases. It is convenient, safe and effective for...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/57C12N1/21C12N9/50C12R1/225
Inventor 钟瑾梁小波还连栋
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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