Mistic融合蛋白促进ω-3脂肪酸去饱和酶Fat-1在大肠埃希菌中高效表达  

Fusion to Mistic improved fatty acid desaturase Fat-1 overexpression in E.coli

作　　者：赵艳 柳欣欣[2] 王大新 李湘鸣[3] 陈平[2] 王昊[2] 

机构地区：[1]扬州大学临床医学院江苏省苏北人民医院医学实验研究中心,江苏扬州225001 [2]扬州大学临床医学院江苏省苏北人民医院普外科,江苏扬州225001 [3]扬州大学医学院,江苏扬州225001

出　　处：《重庆医学》2016年第16期2190-2193,共4页Chongqing medicine

基　　金：国家自然科学基金资助项目(81300721);扬州市科技计划项目(YZ2014204)

摘　　要：目的构建真核膜蛋白ω-3脂肪酸去饱和酶Fat-1基因在大肠埃希菌中的高效表达质粒。方法利用分子克隆技术构建携带Fat-1基因的重组原核表达质粒pET32a-Fat-1和插入膜蛋白表达分子伴侣Mistic基因的pET32a-Mistic-Fat-1;将两种重组质粒转化大肠埃希菌株BL21(DE3),IPTG诱导表达Fat-1蛋白和M110-Fat-1蛋白,通过十二烷基-聚丙烯酰胺凝胶电泳(SDS-PAGE)灰度分析其表达量,蛋白免疫印迹法(Western blot)进一步鉴定蛋白表达。结果经酶切鉴定和测序证实成功构建了pET32a-Fat-1和pET32a-Mistic-Fat-1原核表达载体;SDS-PAGE和Western blot证实ω-3脂肪酸去饱和酶Fat-1在原核表达系统中没有明显诱导表达,而M110-Fat-1融合蛋白在大肠埃希菌中获得了高效表达,占全细胞蛋白总量的15%。结论ω-3脂肪酸去饱和酶Fat-1基因与Mistic基因融合表达,实现了在原核宿主中高效表达真核膜整合蛋白ω-3脂肪酸去饱和酶Fat-1。Objective To construct a highly efficient expression plasmid of eukaryotic nuclear membrane protein Omega 3 fatty acid desaturase gene Fat-1 in E. coli. Methods Using molecular cloning technology to construct the recombinant prokaryotic expression plasmid pET32a Fat-1 and pET32a-Mistic-Fat-1 fused with Membrane proteins expression chaperon mistic;the two recombinant plasmids were transformed into E. coli strain BL21 （DE3）, the expression of Fat-1 protein and M110 Fat-1 protein induced by IPTG were identified by SDS-PAGE and gray degree analysed the amount of expression, further identified by Western blot. Results The results of enzyme digestion and sequencing demonstrated that we successfully constructed the prokaryotic expression vectors pET32a Fat-1 and pET32a-Mistic-Fat-1;SDS-PAGE and Western blot showed that Fat-1 fatty acid desaturase Wasn＇t significantly induced,but the overexpression of M110 Fat-1 fusion protein was obtained in E. coli,accounting for 15% of the total amount of whole cell proteins. Conclusion The fusion with Mistic proteins to express the Fat-1 gene has realized the overexpression of eukaryotic nuclear membrane integrated

关 键 词：Mistic ω-3脂肪酸去饱和酶Fat-1基因 膜蛋白 高效表达 

分 类 号：R378[医药卫生—病原生物学]