| Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are long-chain polyunsaturated fatty acids (LC-PUFAs), both of them belong to ω-3series fatty acids and play an important role on human health. The unique structure of EPA and DHA containing five and six unsaturated bonds, which have a special role and influence on human health. It has confirmed that EPA have an effect on treatment of coronary heart disease, hypertension and inflammation (eg rheumatoid arthritis). DHA is a major element of nervous system cell growth and maintenance and an important composition of the brain and retina, containing up to20%in the human cerebral cortex, the largest proportion accounting for about50%in the retina of the eye, therefore, they have an important effect on the development of mental and visual.The content of ω-3series fatty acids and ω-6series fatty acid in mammals is difference, that is the source of ω-6PUFAs is abundant but that of ω-3PUFAs is very limited, medical research shows that the ratio of ω-6fatty acids and ω-3fatty acid more than a certain content, it may increase the probability of diseases. The ratio of ω-6fatty acids and ω-3fatty acid in the modern Western diet is usually over10, some is up to30. The optimum ratio is4to1or less.The main source of co-3fatty acids are cold water fish in daily diet. At present EPA and DHA in the market are mainly from marine fish oil. But the two content in the fish oil is not stable, because of fish species and season and origin their levels in the fish oil are4%to40%. In addition, the raw materials are expensive,production of fish oil is more complicated and a variety of conditions are complex, so the product is expensive; further more fish oil easily oxidized and with the smell of fish, which make the production and efficacy of EPA and DHA limited.Therefore, through the emerging bio-genetic techniques to obtain a new way of high yield EPA and DHA is a general trend. Now most results of the research is to obtain a higher yield of the EPA, but the yield of DHA is relatively low, because EPA has been confirmed lead to precocious puberty, so obtaining bio-engineering approach of DHA is of important significance. Δ6desaturase and elovl2elongase are two enzymes in the polyene fatty acid metabolic pathway, which can turn EPA into DHA in mammalian cells; the sfatl desaturase which similar to nematode fatl gene has been confirmed can turn AA into EPA, AA are higher levels ω-6series fatty acid in humans and mammals.In this experiment, we first constructed two expression vectors with Δ6desaturase and elovl2elongase, both of the purpose expression vector stably transfected Chinese hamster ovary cells CHO-K1, adding a certain concentration of substrate induction EPA, determining the content of EPA and DHA in cells, thus identification the fuction of Δ6desaturase and elovl2elongase in the way for EPA transform into DHA. Then depend on all the above experiment, we continue to build expression vector with sfatl desaturase, this expression vector and two expression vectors with Δ6desaturase and elovl2elongase were transformed into Chinese hamster ovary cells CHO-K1, adding a certain concentration of substrate induction AA, determination the content of polyunsaturated fatty acids in cells, to verify whether the three genes synergy to promote the AA transform into EPA, and further transform into DHA.The results show that: the fatty acid composition was analysed after join EPA72hours later, compared with the negative control, the content of DHA expressed by Δ6desaturase and elovl2elongase in CHO-K1cells increase by6.57%, the content of EPA decreased by5.70%, indicating that over-expression of the Δ6desaturase and elovl2elongase can enhance the conversion of EPA to DHA in mammalian cells CHO-K1and improve the yield of DHA. The study confirmed the pathway of Δ6desaturase and elovl2elongase promote the conversion of EPA to DHA is feasible, provide a new model use of mammalian cells to produce polyunsaturated fatty acids in the future. |
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