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Method for preparing algae haematochrome through polygenic combination expression and application

An algal red pigment and expression method technology, applied in microorganism-based methods, biochemical equipment and methods, recombinant DNA technology, etc., can solve the problem of high price, achieve easy availability, low cost, suitable for large-scale preparation and application Effect

Active Publication Date: 2014-06-18
CHINA UNIV OF PETROLEUM (EAST CHINA)
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  • Abstract
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Problems solved by technology

[0005] At present, the price of high-purity algae red pigment on the market is relatively high (as a photosensitizer, the price of high-purity algae red pigment is about 150-180 US dollars / mg), and currently it mainly relies on imports from the United States, which is not yet suitable for wide application as a health care product

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  • Method for preparing algae haematochrome through polygenic combination expression and application
  • Method for preparing algae haematochrome through polygenic combination expression and application
  • Method for preparing algae haematochrome through polygenic combination expression and application

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preparation example Construction

[0037] Preparation method of recombinant algae red pigment: clone key enzyme gene of algae red pigment from algae ( hox1 and pebS ),Will hox1 and pebS The gene is cloned into an appropriate expression vector, and then the recombinant expression vector is introduced into the Escherichia coli expression system, and the multi-gene combination expression engineering technology is used to express and produce the recombinant algae red pigment. Finally, through cell collection, chloroform extraction and preparative HPLC chromatography, a red eluate is obtained, that is, the above-mentioned recombinant algae red pigment. The recombinant algae red pigment is a red compound, soluble in dilute salt solution, with a molecular weight of about 586.68g / mol.

[0038]

Embodiment 1

[0040] (1) According to Synechocystis 6803 published in Genebank ( Synechocystis sp. PCC 6803 ) heme oxidase gene ( hox1 ) and Prochlorococcus phage P-SSM2 phycoerythrin synthase gene ( pebS ) gene sequence, design appropriate primers, respectively to Synechocystis 6803 ( Synechocystis sp. PCC 6803 ) and Prochlorococcus phage P-SSM2 genomic DNA as templates, using genetic engineering methods to clone the key enzyme gene of algae red pigment synthesis hox1 and pebS . Algal red pigment synthesis key enzyme gene hox1 and pebS The numbers in Genebank are sll1184 and YP_214290.1.

[0041] (2) Apply multi-gene combination expression engineering method, in hox1 Added to both ends of the gene Bam H I and Sac I restriction site, and inserted into the first multiple cloning site of the pETDuet-1 plasmid, to obtain the recombinant vector pETDuet- hox1 . in gene pebS add at both ends Nde I and xho I restriction site, and cloned into pETDuet- hox1 at anoth...

Embodiment 2

[0044] Pick a single colony of the genetically engineered bacterial strain P1 from the LB solid (LB medium containing 1.5% agar) plate, place it in 5 mL liquid LB medium containing 100 μg / mL ampicillin, and culture it in a constant temperature shaking incubator (temperature: 37°C, speed: about 200r / min). The overnight culture was inoculated into 1 L of TB medium (containing ampicillin 100 μg / mL) at a ratio of 1:100. Cultivate to OD in a constant temperature shaking incubator at a temperature of 37°C and a rotation speed of 200r / min. 600When =0.5~0.7, add isopropyl-β-D-thiogalactopyranoside (IPTG, Isopropyl-β-D-thiogalactopyranoside) to a final concentration of 0.5mmol / L for induction. After adding the inducer, the culture was continued for 24 hours under the culture conditions of temperature 30° C. and rotation speed 200 r / min, and then stopped.

[0045] 10,000 g of the above culture solution was centrifuged for 10 min to collect the bacteria. After the collected bacteria w...

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Abstract

The invention discloses a method for preparing algae haematochrome through polygenic combination expression, and in particular relates to a preparation method for producing the algae haematochrome, which can be applied in preparation of fluoroimmunoassay or functional food, by a polygenic combination expression technology, and application of the algae haematochrome. The preparation method comprises the specific steps of cloning key algae haematochrome synthesis enzyme genes (hoxl and pebS) from spiral seaweeds, and introducing the two key enzyme genes into a recipient cell by the polygenic combination expression technology to build a biological algae haematochrome synthesis way in the recipient cell; converting heme in escherichia coli to synthesize the algae haematochrome through two-step enzymatic catalysis reaction; finally performing extraction and preparative HPLC purification to obtain the high-purity algae haematochrome with the purity over 95 percent. The maximum absorption wavelength of the algae haematochrome is 550nm, and the maximum fluorescence-emission wavelength is 570nm. The algae haematochrome prepared by the method disclosed by the invention can be used as a fluorescence probe or a pigment antioxidant applied in preparation of effective components of the functional food.

Description

technical field [0001] The present invention relates to the fields of genetic engineering and food medicine, in particular to an engineering technology utilizing multi-gene combination expression, in particular to a method for preparing algae red pigment and its application which can be applied in the preparation of immunofluorescent probes or functional foods. Background technique [0002] Algal red pigment is a kind of red algae and blue algae photosynthesis Auxiliary pigments. Similar to bilirubin, it is connected by 4 pyrrole rings in a straight chain through a methylene group, and its molecular weight is about 586.68g / mol. They are bound in the body to a soluble protein called phycoerythrin, which dissolves in dilute salt solutions. They play a role in absorbing and transmitting light energy in photosynthesis. [0003] As a derivative of bilirubin, algal red pigment has been shown to have antioxidative properties (Hirata, T., Tanaka, M., Ooike, M., Tsunomura, T., and...

Claims

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Application Information

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IPC IPC(8): C12P17/16C12N15/70C12N1/21C12R1/19
Inventor 葛保胜王祥法黄方李杰
Owner CHINA UNIV OF PETROLEUM (EAST CHINA)
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