The Cloning And Expression Of Cinoleic Acid Isomerase Gene

CLA (conjugated linoleic acid) is derivate of essential fatty acid LA (linoleic acid), it has the function of anti-tumor, decreasing cholesterol content, anti-oxidation and enhancing immunity capacity as well which the LA doesn't have.It has been proved that LAI ( linoleic acid isomerase) can convert the LA to CLA.Getting LAI and its gene has great significance for the industrial production of CLA.The same strain with Lactobacillus acidophilus was seperated from the fermentation strains agent, and finally indentified it as the NCFM of Lactobacillus acidophilus according to the 16Sr DNA results. Then do the PCR amplification with the primer which is designed according to the sequences of LAI gene published by GENBANK. Then cloned the PCR product into the pMD-19T vector and converted it into E.coli DH5α, and the cloning was indentified right by primary colony PCR, restriction analysis and sequencing. Finally, the LAI gene was subcloned into expression vector pMG36e and intoanspoted into E.coli DH5α, the cloning was indentified right by primary colony PCR and restriction analysis, after that we analyzed its expression.After broking pMG36e-LAI-DH5αstrain, LAI was separated and purificated by salting-out and sephadexG-100 chromatography. Its enzyme activity was assayed and molecular weight of LAI was determinated by SDS-polyacrylamide gel electrophoresis. The results show that the expressed LAI is obtained, which will lay basis for the transporting LAI gene into the strains of lactic acid bacteria and other enetic engineering, in additon, not only will it provide technical support for further production of CLA, but also it will help to the industrial production of high-yield linoleic acid isomerase with high activity.