摘要
通过分析Gen Bank中Burkholderia cepacia脂肪酶的序列,设计简并引物,采用同源克隆的策略,成功地从B.cepacia XYU-6菌株中克隆到脂肪酶基因bcl,其大小为1 095 bp,编码364个氨基酸(Gen Bank登陆号KR233260).将bcl基因与质粒p ET-28b(+)连接并转化大肠杆菌,使脂肪酶BCL的大肠杆菌胞内过表达,其活力是野生菌的12.9倍.通过将bcl基因克隆到细胞表面展示载体p ZXL中,构建脂肪酶BCL的细胞表面展示工程菌,使BCL在Lpp-Omp A引导下定位于大肠杆菌细胞表面,其活力是野生菌的3.9倍.研究结果为脂肪酶BCL后续的分子改造和应用奠定基础.
The lipase gene bcl was isolated from Burkholderia cepacia XYU-6 by homologous cloning method using degenerate primers,which was design based on the analyzing the sequences of lipases from B. cepacia. The gene bcl contained a 1 095 bp open reading frame encoding a protein of 364 amino acids（ Gen Bank accession number：KR233260）. The gene bcl was cloned into the expression vector p ET-28b（ ＋）,and intracellular overexpressed in biologically active in Escherichia coli. Meanwhile,the gene bcl was cloned into the cell-surface display vector p ZXL and transformed into E. coli. The lipase BCL was successfully achieved on the cell surface of engineering strain using the anchoring motif Lpp-Omp A. The two recombinant strains demonstrated 12. 9-fold and 3. 9-fold of lipase activity compared to the wild B. cepacia XYU-6,respectively. This study paves the way for the further research of the lipase for protein engineering and application in the industry.
出处
《江西师范大学学报（自然科学版）》
CAS
北大核心
2015年第5期502-506,共5页
Journal of Jiangxi Normal University(Natural Science Edition)
基金
江西省青年科学基金(20122BAB214011)资助项目
关键词
脂肪酶
克隆
细胞表面展示
表达
lipase
cloning
cell surface display
expression
