双歧杆菌表达载体及启动子的筛选验证

Screening and validation of Bifidobacterium expression vector and promoters

双歧杆菌作为食品级益生菌,其基因工程具有广阔的应用前景和商业价值,但是双歧杆菌的遗传操作系统还不完备,尚处于探索阶段.利用两种载体pLC-nick和pLH01,六种启动子para、pnis、pPRPL、pgap、phup和pHU,采用红色荧光蛋白(red fluorescent protein,RFP)作为报告基因,共构建了12个表达质粒.将重组质粒电转至双歧杆菌中,对于诱导型启动子para加入0.2%(质量分数)L-阿拉伯糖,启动子pnis加入10 mmol/L乳链菌肽诱导RFP蛋白表达,启动子pPRPL则在42℃诱导8 h后表达RFP蛋白.通过检测RFP蛋白的荧光强度,证明在双歧杆菌中利用pLH01载体,phup和pPRPL启动子更有利于RFP蛋白的高效表达.本研究为后续重组双歧杆菌的构建及应用奠定了基础.

Bifidobacterium has been widely used as food-grade probiotics,the genetic engineering of Bifidobacterium has broad application prospects and commercial value.However,the genetic operating system of Bifidobacterium is still in the exploratory stage.In this paper,two expression vectors,pLC-nick and pLH01,as well as six promoters,para,pnis,pPRPL,pgap,phup and pHU were connected to the red fluorescent protein(RFP)gene respectively,a total of twelve recombinant plasmids were constructed.The recombinant plasmids were electrotransformed into Bifidobacterium,0.2%(mass fraction)L-arabinose was added to the inducible promoter para,10 mmol/L nisin was added to the promoter pnis to induce the expression of the RFP protein,and the promoter pPRPL was induced at 42℃for 8 h to express the RFP protein.By detecting the fluorescence intensity of the RFP protein,it was proved that the use of pLH01 vector,phup and pPRPL promoters in Bifidobacterium was more conducive to the high-efficiency expression of the RFP protein.This study laid the foundation for the construction and application of the subsequent recombinant Bifidobacterium.