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Delta 4/ delta 15 fatty acid desaturase in the expression and function of mammalian cells

Author: WangKunFu
Tutor: ZhangTao
School: Jiamusi University
Course: Biochemistry and Molecular Biology
Keywords: ω-3PUFAs Δ4desaturase gene Δ15desaturase gene expression vector DHA EPA
CLC: Q55
Type: Master's thesis
Year: 2013
Downloads: 36
Quote: 0
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Abstract


Objective: Δ4fatty acid desaturase gene sD4which derived from protozoa Euglenagracilis express in the mammalian cell CHO and play its Δ4desaturase activity. With thesynergythe effect of C.Briggsae Δ15desaturase gene sN3, high level of DHA, EPA, and otherω-3PUFAs can be synthesised. In the dual-gene transfected mammalian cells, only need toadd linoleic acid and AA in a medium can be synthesized high level of DHA, EPA and other ω-3PUFAs.Methods: After codon optimized and synthetic of the Euglena gracilisΔ4desaturase genesD4,we connected to the pcDNA3.1(-) vector, while building pcDNA3.1-SN3D4expressionvector. Followed by liposome transfection CHO cells,we use RT-PCR detecting geneexpression at the mRNA level, and detect total fatty acid content of cells by using GC-MS toidentify their functions.Results: Successfully constructed pCDNA3.1-sD4and pCDNA3.1-sN3sD4efficientexpression vector; then successfully transfected these three highly efficient expression vectorinto mammalian cells.The sD4gene was successfully expressed in mammalian cells, showingΔ4desaturation activity that directly convert DPA into DHA. Compared with the control,DHA significantly increase. Meanwhile,sD4gene used the product of sN3gene which using Δ15desaturase accumulated high-level of DPA synthesising a lot of mount of DHA.Conclusion: pCDNA3.1-sD4gene expression vectors and dual-gene expression vectorpCDNA3.1-sN3sD4was constructed successfully and to achieve the high levels of expressionof DHA, EPA in mammalian cells—CHO，that laid a solid foundation for applications oftransgenic animal production and clinical usage and health care industries.

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CLC: > Biological Sciences > Biochemistry > Enzymes
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