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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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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the relevant genes of the important economical cyanobacterium Arthrospira platensis have not been cloned, and its role in the synthesis of fluorescently active phycocyanin is still lacking in research

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
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 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 belongs to the health care product and has application value in food and medicine. It was purchased from the Freshwater Algae Species Bank (FACHB) of the Wild Biology Germplasm Bank of the Chinese Academy of Sciences, and commercially available Arthrospira platensis can also be used.

[0035] Primers hoxI-1 and hoxI-2 were designed, and BamHI and SacI sites were introduced into the primers hoxI-1 and hoxI-2 respectively (underlined enzyme cleavage sites). The primer sequences are:

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

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

[0038] A PCR reaction was performed using the genome sequence of Arthrospira platensis FACHB314 as a template. The PCR reaction procedure was: pre-denaturation at 94°C for 5 min; denaturatio...

Embodiment 2

[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 double-digested with BamHI and SacI and SacI and SalI respectively, and the target fragment was recovered by tapping to obtain the hoxI (314) gene and pcyA ( 314) gene, and connect the two into the pET24a(+) empty vector, first use the enzyme cutting sites BamHI and SacI of the hoxI(314) gene to digest the pET24a(+) empty vector, so that the hoxI( 314) The gene was ligated in first to obtain pET24a-hoxI (314). Then pET24a-hoxI(314) was digested with the enzyme cutting sites SacI and SalI of the pcyA(314) gene, so that the pcyA(314) gene was also connected 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 was screened with a plate containing Kan resistance, and positive strains were obtained th...

Embodiment 3

[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 five constructed recombinant expression strains H(314), P(314), H(314)P(314), H(6803)P(314), H(314)P(6803) into In LB liquid medium (containing the antibiotic Kan); at the same time, inoculate the blank control E.coli BL21(DE3) without expression vector, without antibiotics in the medium, and culture overnight at 37°C for 12-14h;

[0071] (2) Inoculate the overnight cultures of the above five recombinant expression strains in 100ml (containing Kan) LB liquid medium at a ratio of 1:100; inoculated in 100ml LB medium without antibiotics, cultured at 37°C for 2-3 hours to OD 600 ≈0.6;

[0072] (3) At 37°C, add isopropylthio-β-D-galactoside (IPTG) to a working concentration of 1 mmol / L;

[0073] (4) After induction at 37°C for 2 hours, measure the OD of the thr...

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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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