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Delta-6 fatty acid dehydrogenase mutant gene and expression carrier and application thereof

A technology of fatty acid dehydrogenase and expression vector, which is applied in the field of Δ-6 fatty acid dehydrogenase mutant gene and its expression vector and application, and can solve the problems affecting the expression level, etc.

Inactive Publication Date: 2011-11-23
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If the foreign gene contains more host non-preferred codons, especially if they appear continuously, it will seriously affect its expression in the host bacteria

Method used

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  • Delta-6 fatty acid dehydrogenase mutant gene and expression carrier and application thereof
  • Delta-6 fatty acid dehydrogenase mutant gene and expression carrier and application thereof
  • Delta-6 fatty acid dehydrogenase mutant gene and expression carrier and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Preparation of codon-optimized borage fatty acid dehydrogenase eukaryotic expression vector pIRES2-AcGFP1-Delta6

[0037] The nucleotide sequence including EcoRI and XhoI restriction sites, kozak sequence and codon-optimized coding region of borage fatty acid dehydrogenase Delta-6 was synthesized by Invitrogen Company. The backbone vector pIRES2-AcGFP1 (purchased from Clontech) vector was digested with EcoRI / XhoI to obtain the linearized vector sequence; the vector containing the target fragment was also digested with EcoRI / XhoI to obtain the Delta6 gene fragment, and the target fragment was recovered by cutting the gel. 4 DNA ligase was ligated at room temperature for 2 h; the ligated product was transformed into competent cells; the correct new plasmid pIRES2-AcGFP1-Delta6 ( figure 1 ).

Embodiment 2

[0038] Embodiment 2 codon-optimized borage Delta6 gene transfection of HEK293 cells

[0039] The day before transfection, in a 24-well plate at 1.5 × 10 5 HEK293 (purchased from ATCC) (0.5 mL / well) was inoculated at a ratio of 1 / well, and the cell culture medium was replaced with antibiotic-free medium after the cell density reached 90%-95% the next day.

[0040] Dilute 0.8-1.2 μg of pIRES2-AcGFP1-Delta6 plasmid DNA into 50 μL of serum-free OPTI-MEM; dilute 2 μL of liposomes into 50 μL of OPTI-MEM. After 5 minutes, the two were mixed and allowed to stand at room temperature for 20 minutes to form DNA-liposome complexes. Then add the formed DNA-liposome complex into the culture well and mix gently. Place the culture plate at 37°C, 5% CO 2 cultured in an incubator. 4-6 hours after transfection, replace with a new complete medium containing 10% fetal calf serum.

[0041] 24 hours after transfection, the cells were diluted at a ratio of at least 1:10 and passaged, and 0.7 mg / ...

Embodiment 3

[0042] RT-PCR detection of embodiment 3GFP positive cells

[0043] Total RNA of GFP-positive cells was extracted according to the instructions of Trizol (Invitrogen).

[0044] Follow the steps below to synthesize cDNA first strand by reverse transcription:

[0045] 1) DNase (Promega) to treat RNA samples:

[0046] A. Add the reaction system shown in Table 1 to the RNase-free PCR tube:

[0047] Table 1

[0048] Total RNA dissolved in DEPC water

8.0μl

RNase-free DNase 10× buffer

1.0μl

RNase-free DNase

1.0μl

overall system

10.0μl

[0049] B. React at 37°C for 30 minutes;

[0050] C. Add 1.0 μl DNase Stop Solution to stop the reaction;

[0051] D. Inactivate DNase at 65°C for 10 minutes.

[0052] 2) reverse transcription

[0053] A. Add the reaction system shown in Table 2 to the RNase-free PCR tube:

[0054] Table 2

[0055] DNase-treated RNA samples

2.0μl

Primers (gene-specific primers or Oligo dT)...

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Abstract

The invention discloses a delta-6 fatty acid dehydrogenase mutant gene and an expression carrier and application thereof. A codon of a delta-6 fatty acid dehydrogenase mutant gene from borago officinalis is modified into a codon with high or moderate use frequency according to a codon preference of human beings under the condition of not changing an amino acid sequence; furthermore, a kozak sequence is added at an upper stream of an initial codon ATG, and an optimized mutant gene is represented by SEQIDNO: 1. The mutant gene provided by the invention is introduced into a host cell, the fatty acid component analysis is performed on an obtained recombinant host cell strain through a gas chromatography under a condition of adding a substrate linoleic acid. A result shows that a gamma-linolenic acid level in the recombinant host cell is obviously increased; the delta-6 fatty acid dehydrogenase mutant gene provided by the invention can be applied to the increase or improvement of the gamma-linolenic acid content in mammal cells.

Description

technical field [0001] The present invention relates to a fatty acid dehydrogenase mutant gene and its application, in particular to a delta-6 fatty acid dehydrogenase mutant gene derived from borage (Borage officinalis) after codon optimization of the delta-6 fatty acid dehydrogenase gene, The expression vector containing the mutant gene and the host cell containing the expression vector, the present invention also relates to their application in the preparation or transformation of gamma-linolenic acid (GLA), which belongs to the field of delta-6 fatty acid dehydrogenase mutant gene and its application . Background technique [0002] γ-linolenic acid (γ-linolenic acid, GLA, 18:3) is an n-6 polyunsaturated fatty acid with a wide range of biological functions, which is related to a variety of physiological processes affecting health and chronic diseases. Dietary supplementation of γ-linolenic acid (GLA, 18:3 n-6) was found to inhibit smooth muscle cell proliferation in vivo...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/63C12N15/85C12N5/10C12N9/02
Inventor 陈青曹文广
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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