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Spirulina phycobiliprotein alpha subunit total protein gene and encoding protein thereof

A technology of phycobiliprotein and spirulina, which is applied in the field of genetic engineering, can solve the problems that ordinary consumers cannot afford, hinder market expansion, and cumbersome steps, so as to promote high and stable production, have obvious ecological and environmental benefits, and avoid the limitation of raw materials Effect

Inactive Publication Date: 2010-10-27
ZHEJIANG GONGSHANG UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, phycobiliproteins are mainly obtained through separation and purification methods. The steps are cumbersome, the yield is low, and the waste of raw materials is serious. In addition, the conditions for Spirulina cultivation are strict, so the price is expensive and unaffordable for ordinary consumers, which seriously hinders market expansion, research and development. New preparation methods have become urgent

Method used

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  • Spirulina phycobiliprotein alpha subunit total protein gene and encoding protein thereof
  • Spirulina phycobiliprotein alpha subunit total protein gene and encoding protein thereof
  • Spirulina phycobiliprotein alpha subunit total protein gene and encoding protein thereof

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Effect test

Embodiment 1

[0015] Example 1. Obtaining of Spirulina Phycobiliprotein α Submatrix Plasmid

[0016] The extraction method of Spirulina genomic DNA is as follows: Take 200mg of fresh algae mud into a 5ml sterilized centrifuge tube, freeze and thaw with liquid nitrogen for 2 to 3 times, then add 1.5ml of CTAB extraction buffer [1.5%CTAB] preheated to 65°C , 100mmol / L Tris-HCl (pH 8.0), 1.4mol / L NaCl, 20mmol / LEDTA (pH 8.0), 1% (V / V) β-mercaptoethanol (added before use)], add proteinase K solution to the final The concentration was 200 μg / ml. After suspending and mixing evenly, bathe in water at 65°C for 1 hour, and mix by gently inverting from time to time. Then centrifuge at 10000r / min for 5min to remove impurities such as cell debris, add 1.5ml chloroform / isoamyl alcohol (24:1) to the supernatant, mix well and centrifuge at 12000r / min for 10min at room temperature. The supernatant was added with 150 μl of 3 mol / L NaAc (pH 5.2) and 3 ml of pre-cooled absolute ethanol, placed at -20°C for 1...

Embodiment 2

[0022] Example 2. Construction of Spirulina phycobiliprotein alpha subunit, lyase E expression vector

[0023] Using genomic DNA as a template, the lyase E gene was amplified by PCR. The primer sequences are as follows:

[0024] P3: 5′-CCGAGCTCATGAGTGAACCTAACC-3′

[0025] P4: 5′-CCGTCGACTCAGAGTAAACTATCC-3′

[0026] After the amplified fragment was digested by SacI and SalI, the gene was connected to pETDuet-2 which had been digested by the same double enzymes with Solution I to obtain the expression vector pETDuet-3. See the build process figure 2 .

Embodiment 3

[0027] Example 3. Construction of Spirulina phycobiliprotein alpha subunit, lyase E / F expression vector

[0028] Using genomic DNA as a template, the lyase F gene was amplified by PCR. The primer sequences are as follows:

[0029] P5: 5′-CCGTCGACATGGAGGGTAATAGCGTC-3′

[0030] P6: 5′-TATGCGGCCGCCTAGATTGGGCCGATGTTTTCCAGG-3′

[0031] After the amplified fragment was digested by SalI and NotI, the gene was connected to pETDuet-3 which had been digested by the same double enzymes with Solution I to obtain the expression vector pETDuet-4. See the build process image 3 .

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Abstract

Relating to an Alpha subunit holoprotein gene of phycobiliprotein from Spirulina and an encoding protein thereof as well as a recombinant vector containing the Alpha subunit holoprotein gene of phycobiliprotein from Spirulina, the present invention provides an Alpha subunit holoprotein gene of phycobiliprotein from Spirulina, an encoding Alpha subunit holoprotein of phycobiliprotein from Spirulina and an amino acid sequence thereof as well as a recombinant vector containing the Alpha subunit holoprotein gene of phycobiliprotein from Spirulina and an Escherichia coli expression vector constructed by the recombinant vector. Adopting the modern biotechnology, the present invention clones the Alpha subunit gene of phycobiliprotein from Spirulina from algae mud, constructs the microorganism expression vector and transfers the expression vector into Escherichia coli, which can be easily purified and adapted to large-scale industrialized fermentation production, to construct high secretion type engineering bacteria, thus realizing the high-efficiency, low-cost production of the Alpha subunit holoprotein of phycobiliprotein.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a spirulina phycobiliprotein α subunit whole protein gene, its encoded protein and a recombinant vector containing the spirulina phycobiliprotein α subunit whole protein gene. Background technique [0002] Phycobiliprotein is an important light-harvesting pigment protein in cyanobacteria, red algae, dinoflagellates and cryptophyta. It mainly absorbs light energy at 480-660nm and can efficiently transfer light energy to chlorophyll to enable these organisms to perform photosynthesis . Phycobiliproteins contain alpha (α) and beta (β) subunits, the ratio of the two is 1:1, and each subunit is composed of tetrapyrrole-structured prosthetic phycobilin and phycobiliprotein through a thioether bond to remove the prosthetic group Protein cysteine ​​residues are covalently bound. Phycobilin molecules are indispensable to maintain the activity of phycobiliprotein molecules. Phycobilip...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C07K14/405C12N15/63C12N15/70C12N1/21C12R1/19
Inventor 于平励建荣陆伟宏唐云平
Owner ZHEJIANG GONGSHANG UNIVERSITY
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