瘤胃中干酪乳杆菌的亚油酸异构酶基因的克隆与表达

Cloning and Expression of the Linoleic Acid Isomerase Gene of Lactobacillus casei Isolated from Cattle Rumen

以从黄牛瘤胃中分离到的一株具有将亚油酸转化为c9,t11-共轭亚油酸(conjugated linoleic acid,CLA)的干酪乳杆菌(Lactobacillus casei)Fx为出发菌株,提取其基因组DNA,聚合酶链式反应(polymerase chain reaction,PCR)扩增得到1 700 bp大小的亚油酸异构酶(linoleic acid isomerase,LAI)基因片段,将该基因片段纯化后进行TA克隆,得到重组质粒p UCm-T-LAI,将重组质粒p UCm-T-LAI和表达质粒p ET-Dsb A同时进行双酶切,连接得到重组表达载体p ET-Dsb A-LAI,经PCR鉴定和酶切后,将重组表达载体转化到大肠杆菌BL21中,得到具有LAI活性的重组菌株,能将亚油酸转化为c9,t11-CLA,表明从Lactobacillus casei Fx成功克隆LAI,该研究将有助于深入了解不同瘤胃细菌特异性合成不同CLA异构体的LAI基因差异。

Lactobacillus casei Fx was isolated from cattle rumen for its ability to transform linoleic acid into c9,t11-conjugated linoleic acid(CLA).Genomic DNA was extracted and a1700-bp linoleic acid isomerase(LAI)gene fragmentwas amplified from the strain by PCR.The gene fragment was purified and cloned into the plasmid pUCm-T-LAI to obtainrecombinant plasmid pUCm-T-LAI by TA cloning.The recombinant plasmid and the expression plasmid pET-DsbA weresubjected to double enzyme digestion and ligated to the recombinant expression vector pET-DsbA-LAI.After identificationby PCR and enzymatic digestion,the recombinant expression vector was transformed into E.coli BL21.The recombinantstrain,having LAI activity,possessed the ability to specifically transform linoleic acid into c9,t11-CLA isomer.The resultsshowed that the LAI gene from L.casei Fx was successfully cloned.This work makes it possible to understand the differenceamong the LAI genes in different rumen bacteria.