Expression Of The Near N-terminal Polypeptide Of Linoleic Acid Isomerase In Lactobacillus Plantarum P8and Preparation Of Its Polyclonal Antibody

Conjugated linoleic acid (CLA) was a natural fatty acid with many kinds of beneficial function for human body, such as oxidation resistance, anti-cancer, lower cholesterol functions, linoleic acid isomerase (LAI) could convert linoleic acid into CLA. At present, related research on LAI had become the hot spot. But there had been no commercial antibody which was used in detection of linoleic acid isomerase, testing and further research for linoleic acid isomerase was difficult. Therefore, the research for strong specificity, high activity antibody of linoleic acid isomerase appeared particularly important.In his paper, hydrophilic domain of LAI was predicted by using internet biosoftware. The near N-terminal of linoleic acid isomerase contained numerous hydrophilic amino acids, Based on this hydrophilic domain of LAI and the recombinant prokaryotic expression vector was constructed. In view of LAI near N-terminal polypeptide which containing123amino acids a pair of specific primers was designed. The423bp fragment cDNA was amplified by PCR method and was inserted into prokaryotic expression vector pEASY-E1, than was detected by the colony PCR and sequence analysis. The recombinant plasmid was transformed into E.coli BL21and a15ku fusion protein was expressed after the induction with IPTG which was in agreement with the expected molecular weight. The fusion protein was separated, purificated using Ni2+-NTA agarose and detected by SDS-PAGE and Western-blot. The fusion protein was used as an antigen to immunize rabbits to prepare the polyclonal antibody. The double immunodiffusion showed that this anti-serum was very efficient and had a titer of1:132. This result has laid the foundation for further detection of linoleic acid isomerase, its vigorous development and application to product CLA.