食品科学 ›› 2017, Vol. 38 ›› Issue (6): 1-5.doi: 10.7506/spkx1002-6630-201706001

• 生物工程 •    下一篇

瘤胃中干酪乳杆菌的亚油酸异构酶基因的克隆与表达

刘晓华,李慧美,柯颖笑,张凯强,李响敏,李 欣,付金衡,李海星   

  1. 南昌大学中德联合研究院,食品科学与技术国家重点实验室,江西 南昌 330047
  • 出版日期:2017-03-25 发布日期:2017-03-28
  • 基金资助:
    国家自然科学基金地区科学基金项目(31260373);江西省重点研发计划项目(20161BBF60094)

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

LIU Xiaohua, LI Huimei, KE Yingxiao, ZHANG Kaiqiang, LI Xiangmin, LI Xin, FU Jinheng, LI Haixing   

  1. State Key Laboratory of Food Science and Technology, Sino-German Joint Research Institute, Nanchang University, Nanchang 330047, China
  • Online:2017-03-25 Published:2017-03-28

摘要: 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 chainreaction,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pUCm-T-LAI,将重组质粒pUCm-T-LAI和表达质粒pET-DsbA同时进行双酶切,连接得到重组表达载体pET-DsbA-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。

关键词: 共轭亚油酸, 亚油酸异构酶, 干酪乳杆菌, 克隆, 表达

Abstract: 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 a 1 700-bp linoleic acid isomerase (LAI) gene fragment was amplified from the strain by PCR. The gene fragment was purified and cloned into the plasmid pUCm-T-LAI to obtain recombinant plasmid pUCm-T-LAI by TA cloning. The recombinant plasmid and the expression plasmid pET-DsbA were subjected to double enzyme digestion and ligated to the recombinant expression vector pET-DsbA-LAI. After identification by PCR and enzymatic digestion, the recombinant expression vector was transformed into E. coli BL21. The recombinant strain, having LAI activity, possessed the ability to specifically transform linoleic acid into c9,t11-CLA isomer. The results showed that the LAI gene from L. casei Fx was successfully cloned. This work makes it possible to understand the difference among the LAI genes in different rumen bacteria.

Key words: conjugated linoleic acid, linoleic acid isomerase, Lactobacillus casei, cloning, expression

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