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Method for biologically synthesizing conjugated linoleic acid CLA

A technology for synthesizing fatty acids and fatty acids, which is applied in the field of biosynthesis of conjugated linoleic acid (CLA), which can solve the problems of unclear heterologous expression of SCD enzyme function, unclear SCD enzyme biosynthesis of CLA components, etc.

Inactive Publication Date: 2010-10-20
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are no reports of expressing exogenous SCD genes in animal cells or tissues. We do not know the specific function of heterologously expressed SCD enzymes, nor whether heterologously expressed SCD enzymes can effectively biosynthesize CLA components.

Method used

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  • Method for biologically synthesizing conjugated linoleic acid CLA
  • Method for biologically synthesizing conjugated linoleic acid CLA
  • Method for biologically synthesizing conjugated linoleic acid CLA

Examples

Experimental program
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Embodiment 1

[0038] Example 1, Stable expression of exogenous SCD1 gene in HEK 293 cells

[0039] 1. Construction of eukaryotic gene expression constructs

[0040]Human SCD1 gene clone was purchased from Open Biosystems. The clone contains a cDNA fragment of 1080bp (see GenBank accession number BC062303 for the published DNA sequence). The nucleotide sequence of the cDNA is sequence 1 in the sequence listing, and sequence 1 encodes protein SCD1. The amino acid sequence of the protein is listed in the sequence listing. sequence 2. Design and synthesize PCR primers according to the nucleotide sequence of cDNA, the upstream primer is 5'-CCG GAA TTC CGGGCC ACC ATG GAG CAG AAA CTC ATC TCT GAA GAG GAT CTG ATG CCG GCC CAC TTG CTG-3'; the downstream primer is 5' -GGA ATT CCT CAT CAG CCA CTC TTG TAG TTT CCA TCT CCG-3', use the human SCD1 gene clone as a template, carry out PCR amplification to obtain the PCR product, and then connect the fragment of the PCR product digested by EcoRI to the same ...

Embodiment 2

[0049] Example 2, fatty acid determination and CLA biosynthesis directed by exogenous SCD1 gene

[0050] Respectively from the SCD1 negative cell line and the SCD1 positive cell line obtained by embodiment 1, randomly select 4 strains of SCD1 negative cells (numbering is respectively 1,2,19 and 22) and 2 strains of SCD1 positive cells (numbering is respectively 3 and 34 ) according to 2×10 per ml respectively 5 Cells were inoculated into 100-mm culture dishes (Nunc) at a density of 100-mm culture dishes (Nunc), in which 1 copy of No. 22 SCD1-negative cells was used as a blank control without adding substrate.

[0051] method one:

[0052] Four strains of SCD1-negative cells (numbered 1, 2, 19 and 22, respectively, 2 × 10 5 / ml) and 2 strains of SCD1-positive cells (numbered 3 and 34, 2×10 5 / ml) Normal culture (DMEM medium+10% fetal bovine serum; 37 ℃, 5% CO2) after 12 hours, again in the medium (DMEM medium containing trans octadecenoic acid (VA, Sigma V1131) + 10% fetal ...

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Abstract

The invention discloses a method for biologically synthesizing conjugated linoleic acid CLA. The invention provides a recombinant cell which is obtained in order to transfer a coding gene of SCD1 to a host cell, and the amino acid sequence of the SCD1 is a sequence 2 in a sequence table. The coding gene of the SCD1 is shown in a sequence 1 in the sequence table. The coding gene of the SCD1 is transferred to the host cell through a recombinant expression vector. In the method for biologically synthesizing the conjugated linoleic acid, the conjugated linoleic acid is obtained by culturing the recombinant cell. Experiments prove that: the coding gene of the Stearoyl-CoAdesaturase (SCD) gene sequence is transferred to an HEK 293 cell, a cell line stably expressing the SCD1 gene is obtained through G418 screening, heterologously expressed SCD enzyme has various bioactivities, and the content of conjugated linoleic acid and n-7 fatty acid components is obviously increased (p is less than 0.05).

Description

technical field [0001] The invention relates to a method for biosynthesizing conjugated linoleic acid CLA, in particular to a method for synthesizing fatty acids. Background technique [0002] Among food ingredients from natural sources, CLA (Conjugated linoleic acid) is the only natural substance found to have anticancer activity. CLA is a class of octadecadienoic acid composed of positional and geometric isomers, that is, the general term for diunsaturated fatty acid components. Among them, cis-9 and trans-11 isomer (c9t11-CLA), which is the most common and abundant, has anti-cancer properties, can reduce the incidence of cardiovascular diseases, improve immune function, and resist diabetes; another trans-10 , the content of cis-12 isomer (t10c12-CLA) is low, which is involved in the control of body fat deposition and reduces the probability of obesity, which is also a hot spot of current research. In 2008, the US FDA issued a food license, agreeing that CLA can be used ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/64C12N5/10C12N15/53C12N15/63C12N9/02C12R1/91
Inventor 苟克勉
Owner CHINA AGRI UNIV
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