Construction and Expression of Mammal Expression Vector of Ω-3 Fatty Acid Desaturase Gene Fat-1

Abstract: ω-3 PUFAs be suggest to prevent and palliate the symptom of cancer, tumor and Cadiovascular et al. The unbalance beweenω-3 andω-6 PUFAs in human body may cause some diseases and the physiological function detuning. The source ofω-3PUFAs in the diet is limited, in the surveys recently, the ratio has severity deviate, highly to 25:1, also 30:1. If the content ofω-3 PUFAs in beef was elevated, the value would be raise in nutrition, and the palatability would be ameliorated. This kind of beef will be a good source ofω-3PUFAs. Theω-3 fatty acid desaturase, the expressed product of fat-1 gene in Caenorhabditis elegans, can transform C18 and C20ω-6 PUFAs intoω-3 PUFAs, which has important medical and nutrition value. This research cloned fat-1 gene including its whole exons and construct fat-1 gene expression vector pfat-1245-GFP, then the vector was transfected into bovine fetal fibroblasts and positive cells was selected to check the content ofω-6 andω-3 PUFAs.The fat-1 genomic cDNA was cloned from total RNA of tissues of C.elegans with PCR, which was 1245 bp and comprised intact coding regions. Sequence analysis demonstrated that its sequence with the relative region of fat-1 gene sequence on GenBank was same. Then the objective fragment on fat-1 was cloned directionally into expression vector PIRES2-AcGFP-Nu. As a result, the expression vector for fat-1 was constructed, which included resistant gene (neo) and reporter gene (GFP).The vector Pfat1245-IRES-GFP was transfected into bovine fibroblasts cultured in vitro. The expression of report gene GFP was observed in positive cells derived from G418 selection under fluoroscope. The culture system was amplified to get more cells. The result of RT-PCR showed that the cloned fat-1 gene can expressed primarily in bovine fibroblasts. The vector Pfat1245-IRES-GFP are able to efficiantly express fat-1 gene.Cellular lipids extracts from stably selected cells were analyzed with HPLC and the results showed that amount of totalω-6 PUFAs dropped form 35.60% to 25.47%, whereas the amount of totalω-3 PUFAs increased form 5.75% to 12.33%. Theω-6/ω-3PUFAs ratio also dropped from 6.19 to 2.07, and expression of fat-1 quickly and effectively elevated the cellularω-3 PUFAs contents and improved the ratio ofω-6/ω-3 PUFAs. These data demonstrates the Fat-1 gene can produceω-3 PUFAs by using the correspondingω-6 PUFAs as substrates. The mammal expression vector ofω-3 fatty acid desaturase gene fat-1 was constructed successful….
Key words: ω-3 fatty acid desaturase gene; polyunsaturated fatty acids; mommal espression vector; cattle fibroblasts; Caenorhabditis elegans

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