Construction and Expression of Recombinant Plasmids of n-3 Fatty Acid Desaturase Gene

Abstract: Polyunsaturated fatty acids (PUFAs) are important membrane components and precursors of signaling molecules.Omega-3 polyunsaturated fatty acids are required for normal cellular function and have been shown to exert many preventive and therapeutic actions. But mammals cannot naturally produce omega-3 (n-3) fatty acidsElevatin tissue concentrations of n-3 PUFAs, for mammalian cells lack enzymatic activities necessary either to synthesize the precursor of n-3 PUFAs or to convert n-6 to n-3 PUFAs.In order to analyze the expression of n-3 fatty acid desaturase gene (Mfat-1) in vivo, the experiment was carried out to construct a recombinant plasmid of MFat 1 gene by PCR technique. The enzyme sites of BamHI and PstI were introduced to the C-end, SalI and termination code inserted to the N-end of Mfat-1 gene which was derived from Caenorhabditis elegans gene, sFat1. The PCR amplified product of the fusion gene was doubly digested with BamHI and SalI endonucleases and then cloned into the pGEX-4T-l expression plasmid which was also digested with the BamHI and SalI. The recombinant plasmid was digested with BamHI and SalI to identify whether the sFat1 cDNA fragments were inserted into the plasmid in correct orientation. .The BL21 (DE3) competent cells were transformed with the recombinant pGMFat-1, incubated with 2xYTA culture and induced to express the fusion protein by IPTG. The mRNA and protein expression of the recombinant plasmids were assayed by RT-PCR and SDS-PAGE. The results were found that the mRNA could be correctly transcribed from the fusion gene and the fusion protein was expressed in the form of inclusion body with 46KD molecular weight revealed by SDS-PAGE analysis.Based on the preliminary analysis to the characteristics of the recombinant pGEX-Mfat-1, we constructed the eukaryotic recombinant plasmids to express the Mfat-1 in eukaryotic cell. The Mfat-1 genes of was sub-cloned into eukaryotic nonsecreting vector VR1012 and was identified by digestion with PstI and SalI. The RT-PCR analysis proved that the Mfat-1 gene could be correctly expressed in the eukaryotic cells through transfection, which is of importance for future application of Mfat-1 gene to improve the nutrition quality of animal meat and milk…
Key words: n-3 fatty acid desaturase gene; recombinant plasmid; construction; transfection; expression

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