Gene engineering strain for expressing phycocyanobilin and constructing method and application thereof

A technology of genetically engineered strains and phycocyanin, which is applied in the fields of molecular biology technology and bioengineering, can solve problems such as lack of research on effects, and achieve the effect of broad market application prospects.

Inactive Publication Date: 2017-06-13
OCEAN UNIV OF CHINA
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  • Claims
  • Application Information

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Problems solved by technology

However, the relevant genes of the important economical cyanobacterium Arthrospira platensis have not be

Method used

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  • Gene engineering strain for expressing phycocyanobilin and constructing method and application thereof
  • Gene engineering strain for expressing phycocyanobilin and constructing method and application thereof
  • Gene engineering strain for expressing phycocyanobilin and constructing method and application thereof

Examples

Experimental program
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Effect test

Example Embodiment

[0032] Example 1: Cloning of hoxI gene and pcyA gene in Arthrospira platensis FACHB314

[0033] 1. Cloning of hoxI gene in Arthrospira platensis FACHB314

[0034] Arthrospira platensis FACHB314 is a health care product with application value in food and medicine. It is purchased from the Freshwater Algae Species Bank (FACHB) of the Wildlife Genebank of the Chinese Academy of Sciences. Commercially available Arthrospira can also be used.

[0035] Design primers hoxI-1 and hoxI-2, and introduce BamHI and SacI sites on the primers hoxI-1 and hoxI-2, respectively (the underline is the restriction site). The primer sequences are:

[0036] hoxI-1 5'- GGATCC ATGAGTGTTAACCTAGCCACCCA-3';

[0037] hoxI-2 5'- GAGCTC TTAGTTCACCGAGTCAGTAGCG-3'.

[0038] The PCR reaction was carried out with the genome sequence of Arthrospira platensis FACHB314 as a template. The PCR reaction procedure: 94°C pre-denaturation for 5min; 94°C denaturation for 30s, 55°C annealing for 30s, 72°C extension for 1min, 30 cy...

Example Embodiment

[0051] Example 2: Establishment of recombinant expression strain H(314)P(314)

[0052] The pMD19T-hox I (314) and pMD19T-pcyA (314) of the obtained positive clone plasmids were digested with BamHI and SacI and SacI and SalI respectively, and the target fragments were recovered by tapping to obtain hoxI (314) gene and pcyA ( 314) gene, and ligate the two into the pET24a(+) empty vector. When ligating, first use the restriction sites BamHI and SacI of the hoxI(314) gene to cut the pET24a(+) empty vector, thereby cutting the hoxI(+) empty vector. 314) The gene is first ligated to obtain pET24a-hoxI (314). Then pET24a-hoxI(314) was digested with the restriction sites SacI and SalI of the pcyA(314) gene, so that the pcyA(314) gene was also ligated into the expression vector to obtain the recombinant expression plasmid pET24a-hoxI(314) -pcyA(314).

[0053] The plasmid was transformed into E.coli DH5α, and screened with Kan-resistant plates, and the positive strains were identified by P...

Example Embodiment

[0068] Example 3: Induced expression and SDS-PAGE of recombinant expression strains H(314), P(314), H(314)P(314), H(6803)P(314), H(314)P(6803)

[0069] 1. Induced expression of recombinant strains:

[0070] (1) Inoculate the constructed 5 recombinant expression strains H(314), P(314), H(314)P(314), H(6803)P(314), H(314)P(6803) respectively In LB liquid medium (containing antibiotic Kan); At the same time, inoculate blank control E.coli BL21(DE3) without expression vector, the medium does not contain antibiotics, and cultivate overnight at 37℃ for 12-14h;

[0071] (2) Inoculate the overnight cultures of the above five recombinant expression strains in 100ml (including Kan) LB liquid medium at a ratio of 1:100; the overnight cultures of blank E. coli BL21 (DE3) at a ratio of 1:100 Inoculate 100ml of LB medium without antibiotics in the proportion of, and incubate at 37℃ for 2-3 hours to OD 600 ≈0.6;

[0072] (3) At 37°C, add isopropylthio-β-D-galactoside (IPTG) to a working concentrati...

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Abstract

The invention provides gene engineering strain for expressing phycocyanobilin and a constructing method and application thereof. The constructing method disclosed by the invention comprises the following steps of cloning ferroheme oxidase hoxI gene and phycocyanobilin ferreoxin reductse gene pcyA of arthrospira platensis, connecting into a pET24a(+) expression vector, constructing out a recombinant expression plasmid pET24a-hoxI(314)-pcyA(314), converting into E.coli BL21(DE3) expression strain and obtaining recombinant expression strain H(314)P(314). Therefore, the constructing method achieves the purpose that all elements of Escherichia coli heterologous expressed phycocyanobilin are all from the arthrospira platensis. The Escherichia coli heterologous expressed phycocyanobilin can be further combined with alpha and beta subunits of heterologous expressed phycocyanin, so that biosynthesis of phycocyanin with fluorescent activity can be catalyzed. The phycocyanin extracted from the arthrospira platensis can serve as natural pigment to be applied to the fields of food, cosmetics and the like.

Description

technical field [0001] The invention belongs to the fields of molecular biology technology and bioengineering technology, and specifically relates to a genetically engineered bacterial strain expressing phycocyanin and its construction method and application. Background technique [0002] Phycobiliproteins mainly exist in red algae, cyanobacteria, cryptophyta and a few dinoflagellates, composed of apoproteins and chromophores, and are pigment protein complexes that can capture blue-green light that higher plants cannot use. At the same time, phycobiliproteins have multiple functions and can be used as fluorescent probes, food additives, health products and medicines. According to the differences in the absorption spectrum properties of phycobiliproteins, they can be divided into four categories: phycoerythrin, phycoerythrin, phycocyanin, and allophycocyanin. Optically active phycobiliproteins are mainly composed of apophycobiliproteins and phycobilin covalently bonded. Phy...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/66C12P17/16C12P21/02C12R1/19
CPCC12N15/70C07K14/795C12N9/0083C12N9/88C12N15/66C12N2800/101C12P17/165C12Y114/99003
Inventor 臧晓南肖东芳张学成徐涤吴非张冉冯小亭衣俊杰
Owner OCEAN UNIV OF CHINA
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