Expression Of Delta5Fatty Acid Desaturase From Mortierella Isabellina High-yield Mutant Strains In Escheirchia Coli

Arachidonic acid is an important ω-6polyunsaturated fatty acids.It is important in maintainingthe structure and function of cell membrane.It not only as a kind of very important structure lipidwidely exists in the mammalian tissue organs (especially nervous tissue),but also many20carbonderivatives such as prostaglandin E2, top ring, silane A2and leukotrienes thrombus precursorsubstances.It has a wide range of biological activity and important role in nutrition.ARA in the bodycan’t synthesis, must by food supply their corresponding precursors such as fatty acids—linoleic acidand α-linolenic acid, and on this basis through dehydrogenation and extend the role of carbonchain.Δ5-fatty acid dehydrogenase catalyzed is the enzyme of ARA form the last step of thedesaturation reaction, It is two high-γlinolenic acid as substrate, catalytic dehydrogenation to formits fifth carbon ARA,It is speed limit of enzymes in ARA synthetic way.The primers were designed according to the sequences of△5-fatty acid desaturase gene in theGenBank and the multipleclone sites of the sequence of pET-30a(+)vector.Extract the genome RNAof Lactobacillus plantarum and amplify△5fatty acid desaturase gene from Lactobacillus by PCRand clone into expression vector pMD18-T,transform the constructed recombinant plasmid to E.coliDH5α,through PCR,double digesting,T4-ligase Iigating and potsereening,the fragment of△5-fattyacid desaturase gene was subcloned into the pET30a(+).The idenitified plasmid was transferred intoE.coli BL21(DE3)under induction of IPTG.In IPTG concentration1.0tendency for7h L, induced thehighest expression level, expresses the55kda protein (including six histidine label), and proteinpurification, sds-page electrophoresis, finally by the Western bloting appraisal, the results show thatthe protein can be5fatty acid dehydrogenase specific reaction to delta, have good antigenicity,confirmed that the protein is delta5fatty acid dehydrogenase.This study cloned the dark yellow is spore mould delta5fatty acid dehydrogenase gene,recombinant expression plasmid was constructed successfully, obtained stable expression pET30(+)escherichia coli prokaryotic expression system, which for delta5fatty acid dehydrogenase structureand related function study and constructing gene engineering strains laid a certain foundation.