Construction Of A Prokaryotic Expression Vector For Linoleic Acid Isomerase Gene And Induced Expression

Linoleic acid isomerase could catalyze linoleic acid into conjugated linoleic acid. Agrowing number of scholars are interested in it for the advantages of the high conversion rate,production of high purity of CLA, mild reaction conditions, and so on. In order to improve theyield of linoleate isomerase,the gene of linoleic acid isomerase are cloning, and reorganizated,expressed in Escherichia coli. However, due to the defects of E. coli expression system,recombinant protein is inactive inclusion bodies, which undergoes a complex series ofdenaturation, renaturation process in order to restore its natural structure and biologicalactivity, that increases the cost of the experiment. We choses Bacillus subtilis expressionsystem in this topic to induced express the restructuring of LAI, hoping to solve the aboveproblem.Based on Genbank submission sequences, primers are designed by the PerlPrimersoftware. Linoleic acid isomerase gene from Lactobacillus acidophilus and Lactobacillusplantarum is amplified separately, which templates are Lactobacillus plantarum genomes andLactobacillus acidophilus plasmid. Then, the fragments are connected to pMD-19T vectorand transformed into Escherichia coli DH5α, sequencing after double enzyme digestion andPCR identification. Blasting the sequencing results and analysising bioinformatics featuresusing biological software. We judge preliminary that the nature of the clone sequences arecloser to the original and name the cloning vector as LA-T、LP-T.pSBPTQ, a Bacillus subtilis expression vectors of inducible secretion, is selected in thiswork. The cloning vector and expression vector are digested by XbaⅠand KpnⅠovernight.Then the products are recoveried and connected with Solution Ⅰ, Escherichia coli DH5αatlast. Naming the new vector as LA-pSBPTQ. The new expression vector are transformed intoBacillus subtilis DB1342after double enzyme digestion and PCR identification. According tothe future of plasmid pSBPTQ, the new vector is induced culture still by sucrose.56hourslater, centrifuge the supernatants of fermented liquor, taking supernatant and supernatantliquid concentrated respectively for SDS-PAGE detection,we detecte the linoleic acidisomerase activity of supernatant choosing UV spectrophotometer method. The experimentalresults show that the new vector secretes linoleic acid isomerase to the extracellular andsecretion of linoleic acid isomerase is activity in transgenic engineering bacterium Bacillussubtilis.