乳酸菌红色荧光蛋白融合表达系统的构建  

Establishment of Red Fluorescent Protein Fusion Expression System of Lactic Acid Bacteria

作　　者：寇田田 北婷婷 李晨[1,2] 许沙[1,2] 田洪涛[1,2] 罗云波[3] Kou Tiantian Bei Tingting Li Chen Xu Sha Tian Hongtao Luo Yunbo(College cf Food Science and Technology, Agricultural University of Hebei, Baoding 071001, Hebei Agricultural Products Processing Engineering Technology Research Center of Hebei, Baoding 071001, Hebei College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083)

机构地区：[1]河北农业大学食品科技学院,河北保定071001 [2]河北省农产品加工工程技术研究中心,河北保定071001 [3]中国农业大学食品科学与营养工程学院,北京100083

出　　处：《中国食品学报》2017年第3期235-240,共6页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家自然科学基金项目(31301520);河北省自然科学基金项目(C2016204129);国家"863"计划项目(2006AA10Z317)

摘　　要：目的:构建以红色荧光蛋白基因(dsred2)为标记的乳酸菌融合表达系统p MG36e-dsred2,并以α-淀粉酶(amy)为报告基因实现融合基因dsred2-amy在乳酸菌内的融合表达。方法 :通过PCR的方法以质粒p PIC9kdsred2为模板扩增得到dsred2基因的全部编码区序列,并将其连接到克隆型载体p MG36e上,得到重组载体p MG36e-dsred2。以地衣芽孢杆菌基因组为模板,扩增只带有起始密码子而不带有终止密码子的α-淀粉酶基因(amy),将其连接到克隆型载体p MG36e-dsred2上,获得融合表达载体p MG36e-dsred2-amy。最后将该载体电转化干酪乳杆菌,利用荧光显微镜观察红色荧光基因表达情况,用淀粉平板检测淀粉酶活性。结果:通过PCR的方法分别扩增到大小为694bp的红色荧光蛋白基因(dsred2)和大小为1 554 bp的淀粉酶基因(amy)。将获得的两个基因片段进行测序,结果与NCBI数据库进行对比,dsred2基因的匹配率为100%,amy基因同源性为99.54%。成功构建了乳酸菌表达系统p MG36e-dsred2及融合表达载体p MG36e-dsred2-amy。在荧光显微镜下观察菌体有荧光且淀粉平板上有明显的透明圈。结论:融合表达载体的构建成功为乳酸菌在生物体内的定植、分布、存活等研究提供了方法,也为研究外源蛋白在乳酸菌内的表达的分泌(细胞内、细胞壁、细胞外表达)、活性检测等奠定了基础。The aim of this study was to construct lactic acid bacteria filsion expression system pMG36e-dsred2 using red fluorescent protein gene （dsred2）, and to achieve the dsred2-amy fusion gene expression. Methods： The ORF of dsred2 gene was amplified with pPIC9k-dsred2 as template, and was cloned in the vector pMG36e to construct the recombinant vector pMG36e-dsred2. The alpha amylase gene without the termination codon was amplified with Bacillus licheniformis genome as template and was cloned in the vector pMG36e-dsred2 to construct the fusion expression vector pMG36e-dsred2-amy. Finally, the plasmid was eleetroporated into Lactobacillus casei, and we detected the red fluorescent gene expression with fluorescence microscopy and amylase activity with starch plate. Results： The red fluurescent protein gene （dsred2） with size of 694bp and the the amylase gene （amy） with size of 1 554 bp were amplified, and both of the gene fragments were sequenced and compared with the NCBI database. The homology of dsred2 and amy gene was 100% and 99.54% respectively. The bacteria was detected with fluorescence and a transparent circle on the plate Conclusion： The construction of fusion expression vector supplied a new method to analyze the colonization, distribution and survive of lactia acid bactdfia in vivo. In addition, the research achievement lay the foundation of revealing the secretory expression condition （in the cell, on the cell wall or the out of the cell） of laetia acid bacteria and detecting the activity of heterologous protein.

关 键 词：乳酸菌 融合表达载体 红色荧光蛋白 淀粉酶 

分 类 号：TS201.3[轻工技术与工程—食品科学]