Heterologous Expression And Polymorphism Analysis Of Linoleic Acid Isomerase Of Lactic Acid Bacteria In Rumen

Conjugated linoleic acid(CLA) is generally referred to as a series of geometric and positional linoleic acid isomer which have cis or trans conjugated double bond.It has a wealth of nutritional value,different isomers also have different physiological functions, such as c9,t11-CLA has the effect of inhibiting tumor, while t10,c12-CLA is able to prevent diabetes and lose weight. In this study, four strains of lactic acid bacteria, which have linoleic acid isomerase activity, were screened from cattle rumen. After the LAI gene was amplified from four strains, the prokaryotic engineering bacterias were reconstructed and the polymorphism of the LAI gene was analyzed by PCR-RFLP technique. The experimental results are as follows:Firstly, construction and expression of LAI prokaryotic engineering bacteria:four strains of lactic acid bacteria with the ability of converting LA to CLA were isolated from the cattle rumen. Employing the genomic DNA of lactic acid bacteria as the template of PCR, then subcloning the PCR product into the pUCm-T vector,we sucessfully finished the endonuclease analysis and the cloning procedure after sequencing the coding region. Furthermore, we cut the above PCR product and pET-DsbA with the same restriction enzyme, with the purpose of getting the engineering strains of pET-DsbA-LAI. The recombinant expression vectors were transformed into BL21. Addition of IPTG induced the expression of these four recombinant strains. The results of SDS-PAGE electrophoresis showed that the recombinant strains expressed the fusion protein which size is about 85 kDa(LAI:64,DsbA:21k Da).Secondly, optimization of induction conditions of recombinant strains: single factor experiment was carried out on the induction temperature, induction occasion,induction time and inducer concentration of the recombinant strain.The optimal expression conditions of recombinant strain BL21(pET-DsbA-206-LAI) were gotten:induced occasion for the training of 7 h after induction; induction time of 15 hours;induced by the temperature of 30?; induced IPTG at a final concentration of 100?g/mL. The optimal expression conditions of recombinant strainBL21(pET-DsbA-Fx-LAI) were gotten: induced occasion for the training of 10 h after induction; induction time of 15 hours; induced by the temperature of 25 ?;induced IPTG at a final concentration of 100 ?g/mL.Finally, polymorphism analysis of linoleic acid isomerase gene: linoleic acid isomerase gene of four strains were compared, and found that the similarity of LAI gene of two Lactobacillus plantarum reached 99.5%, and the similarity of LAI gene of two Lactobacillus casei reached 99.8%. But the similarity of the Lactobacillus plantarum and Lactobacillus casei is only 40.9%.Using Nde?, Dra?, Eco R? for single enzyme digestion of LAI genes, respectively. The results show that Lactobacillus plantarum has two Nde? enzyme site while Lactobacillus casei only had one. Lactobacillus plantarum and Lactobacillus casei all had one Dra ?enzyme site. While Lactobacillus plantarum had no Eco R ? enzyme site,Lactobacillus casei had two sites. These two results have proved that the linoleic acid isomerase gene of different species of strains exist great differences.