The Cloning Of C. Elegans Fat-2Gene And Expression In The Ear Fibroblast Cells

ω-6polyunsaturated fatty acids (ω-6PUFAs) which is an important component of cells, and human essential fatty acids, has been subject to wide attention. The study found that the co-6PUFAs plays a vital role in the reduction and prevention of cardiovascular and cerebrovascular diseases, cancer and other diseases. But mammals lack of synthesis of ω-6PUFAs dehydrogenase gene using genetic engineering to change this situation made possible with advances in technology. In this experiments, the healthy Simmental ears and the nematode C. elegans, are builted in ear fibroblast cells as experimental materia-1nematodes of C.elegans the fat-2gene eukaryotic expression vector, and make it final in ear fibroblast cells expressions will follow-up of genetically modified to provide help, the next stage of Simmental crossbred cattle construction oftransgenic embryos and laid an important foundation.The experiments eultured cow fibroblasts sueeessfully through tissue culture method. The whole CDS sequence of C. elegans Fat-2gene was cloned by PCR and bioinf-ormatics analysis results showed that the nematodes of Fat-2gene CDS sequence was1276bp and code454amino acids;the coding protein (43.46Kd molecular weight and6.73isoelectric point) have four transmembrane domains that three areas are located at the membrane, and that is a transmembrane protein.Experiments with pEGFP-N eukaryotic expression vector as the backbone, usin-g theEcoRI and Smal restriction enzyme, was successfully constructed recombinant eukar-yotic expression vector pEGFP-NI-fat-2, and transfected to become the leader of the fibroblasts. G418selection for14days to get the integration of target gene-positive cell lines. Take transfected cell lysis to release the protein were analyzed by Westernblot and gained a43KD stripe which fitted well with the theoretieal value of the produet translated by fat-2gene, which demonstrated that the fat-2gene had expressed in ear fibroblasts cells.