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A method and application of multi-gene combination expression to prepare algae red pigment

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

Active Publication Date: 2016-05-18
CHINA UNIV OF PETROLEUM (EAST CHINA)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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

Method used

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  • A method and application of multi-gene combination expression to prepare algae red pigment
  • A method and application of multi-gene combination expression to prepare algae red pigment
  • A method and application of multi-gene combination expression to prepare algae red pigment

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Experimental program
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Effect test

Embodiment 1

[0038] (1) According to the gene sequences of Synechocystis sp. PCC6803 heme oxidase gene (hox1) and ProchlorococcusphageP-SSM2 phycoerythrin synthase gene (pebS) published in Genebank, appropriate primers were designed, respectively Algae 6803 (Synechocystissp. PCC6803) and ProchlorococcusphageP-SSM2 genomic DNA were used as templates, and the key enzyme genes hox1 and pebS of algae red pigment synthesis were cloned by genetic engineering methods. The numbers of the key enzyme genes hox1 and pebS in algae red pigment synthesis in Genebank are sll1184 and YP_214290.1, respectively.

[0039] (2) Using the multi-gene combination expression engineering method, add BamHI and SacI restriction sites at both ends of the hox1 gene, and insert them into the first multiple cloning site of the pETDuet-1 plasmid to obtain the recombinant vector pETDuet-hox1 . Add NdeI and XhoI restriction sites at both ends of the gene pebS, and clone into another multiple cloning site of pETDuet-hox1 (s...

Embodiment 2

[0042] Pick a single colony of the genetically engineered strain P1 from the LB solid (LB medium containing 1.5% agar) plate, put it in 5 mL liquid LB medium containing 100 μg / mL ampicillin, and culture it in a constant temperature shaking incubator (temperature: 37 ℃, speed: about 200r / min). The overnight culture was inoculated in 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. 600 When =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.

[0043] 10,000 g of the above culture solution was centrifuged for 10 min to collect the bacteria. After the collected bacteria were washed tw...

Embodiment 3

[0048] Primers were designed according to the hox1 and pebS gene sequences published in Genebank. Hox1 forward primer P1: 5'-TA CATATG TATGAGTGTCAACTTAGCTTCCCAG-3', reverse primer P2: 5'-TT GATATCCTAGCCTTCGGAGGTGGCGAG-3'. Forward primer P3 of pebS: 5'-TT GATATC ATGACGAAGAACCCGCG-3', reverse primer P4:5'-TG CTCGAG TTACTTGTAGGAGAACAG-3'. Genomic DNA of Synechocystissp.PCC6803 and ProchlorococcusphageP-SSM2 were used as PCR amplification templates respectively. The desired gene fragment was amplified by PCR. PCR products were purified by Cyclepure kit (Omega Company). First, the obtained hox1 gene fragment and the pET28a vector were respectively digested with corresponding endonucleases at 37° C. for 6 h. The digested product was recovered from the gel after agarose gel electrophoresis, the gene fragment was mixed with the carrier at 3:1, ligated overnight at 16°C under the action of T4 DNA ligase, and the ligated product was transformed into Escherichia coli DH5α. Sc...

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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, algae red pigment has been shown to have anti-oxidation (Hirata, T., Tanaka, M., Ooike, M., Tsunomura, T., and Sakaguchi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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