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Recombinant bacillus subtilis expressing C30 carotenoid

A technology of Bacillus subtilis and carotene, applied in the field of biotechnology and genetic engineering, to achieve the effect of reducing the symptoms of colitis

Active Publication Date: 2016-08-10
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

But this enzyme has not been identified in bacteria

Method used

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  • Recombinant bacillus subtilis expressing C30 carotenoid
  • Recombinant bacillus subtilis expressing C30 carotenoid
  • Recombinant bacillus subtilis expressing C30 carotenoid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0083] 1 Construction of carotenoid synthase plasmid pMK3-crtmn

[0084] The methods used in the following examples are conventional methods unless otherwise specified. The specific materials and reagents involved are as follows:

[0085] Bacillus subtilis strain WB800 was donated by Gao Xuewen, School of Plant Protection, Nanjing Agricultural University. For literature, see: Wu, H., Wang, S., Qiao, J., Liu, J., Zhan, J., Gao, X., 2009. Expression of HpaG Xooc proteinin Bacillus subtilis and its biological functions. Journal of Microbiology and Biotechnology 19, 194-203;

[0086] E.coli JM109, plasmids pMK3, pCDNA3.1 (purchased from Life Technologies);

[0087] Reagents: Q5 high-fidelity enzyme was purchased from NEB Company; pMD19-T vector, Agarose Gel DNA Purification Kit, restriction endonuclease, T4 ligase, etc. were purchased from Takara Company; kanamycin sulfate, ampicillin, bacterial genome extraction reagents The kit was purchased from OMEGA; the One Step Cloning...

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Abstract

The invention relates to recombinant bacillus subtilis capable of synthesizing carotenoid 4,4'-diaponeurosporene with 30 carbon atoms (C30), and belongs to the biotechnology genetic engineering field. A bacillus subtilis expression plasmid pMK3-crtmn (which is capable of expressing C30 carotenoid synthases Crtm and Crtn in bacillus subtilis) containing C30 carotenoid synthase genes crtm and crtn is successfully constructed, and is successfully electro-transformed into bacillus subtilis WB800. The WB800 strain after transformation turns from the original color of white to yellow. A pigment component extracted from the recombinant WB800 is identified as 4,4'-diaponeurosporene by high performance liquid chromatography (HPLC). 4,4'-diaponeurosporene can stimulate maturation of dendritic cells, and generated cytokines (IL-6, IL-10, IL-12p70 and TNF[alpha]) have the amount increased, and moreover and have stronger ability to stimulate proliferation of T lymphocytes. With oral administration of the bacillus subtilis capable of synthesizing 4,4'-diaponeurosporene, the dextran sulphate sodium (DSS)-induced mice colitis symptoms can be significantly reduced. The bacillus subtilis capable of producing 4,4'-diaponeurosporene is expected to be developed as probiotics for prevention of colitis.

Description

1. Technical field [0001] The present invention relates to the expression of C 30 The invention relates to carotenoid recombinant bacillus subtilis, which belongs to the field of biotechnology genetic engineering. Specifically include: C 30 Construction of carotenoid expression plasmid pMK3-crtmn; C 30 Biosynthesis and identification of carotenoids; C 30 Carotenoids can stimulate the maturation of dendritic cells, increase the amount of cytokines (IL-6, IL-10, IL-12p70 and TNFα) produced, and have a stronger ability to stimulate the proliferation of T lymphocytes; oral administration can express C 30 Bacillus subtilis carotenoids can significantly reduce dextran sodium sulfate (DSS)-induced colitis symptoms in mice. 2. Background technology [0002] 1. Research progress on carotenoid synthesis [0003] Carotenoids are a class of substances that widely exist in nature, many of which are essential nutrients for humans and animals. Carotenoids in nature come from two carb...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N1/21C12Q1/02A61K35/742A61P1/00C12R1/125
CPCA61K35/742C12N9/00C12N15/75C12N2800/101G01N33/5038G01N33/5047
Inventor 杨倩刘浩飞徐文雯朱立麒
Owner NANJING AGRICULTURAL UNIVERSITY
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