摘要
为构建乳酸菌Nisin诱导表达载体,实现外源目的基因在乳酸菌中的可控表达,本研究以产Nisin乳酸乳球菌染色体DNA为模板,采用PCR技术扩增双组份调控元件nisRK基因和诱导型启动子nisA基因,并克隆至乳酸菌-大肠杆菌穿梭质粒pW425et中,以nisA基因替换原组成型启动子P32,构建乳酸菌Nisin诱导表达载体pW425N。为检测载体的诱导表达功能,以gfp基因作为报告基因,构建重组表达载体pW425N-gfp,以分离自仔猪肠道内嗜酸乳杆菌为受体菌,电转化法制备重组乳酸菌pW425N-gfp/L.acidophilus。结果表明,重组载体能够在Nisin诱导下表达目的荧光蛋白,并且最佳Nisin有效浓度为30 ng/mL,最佳诱导时间为3 h。该表达载体的构建为外源功能性蛋白在乳酸菌中的诱导表达奠定基础。
To construct Nisin-controlled expressive plasmid for Lactococcus lactis, the two-component of the regulatory element nisRK gene and inducible promoter nisA sequence were amplified by PCR from the chromosome DNA of Nisin producing L. lactis. The nisRK was inserted into the E. coli-L, lactis shuttle plasmid pW425et and the constitutive promoter P32 was replaced by inducible promoter nisA to construct Nisin-controlled expression plasmid pW425N with gfp as a report gene. The resultant recombinant plasmid of pW425N-gfp was eleetroporately transformed into L. acidophilus which isolated from piglet intestine gut. The results showed that the GFP protein was able to be efficiently expressed by Nisin induction at 30 ng/mL for 3 hours.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2012年第8期624-628,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金项目(81170358)
吉林省世行贷款农产品质量安全项目(2011-Y07)
吉林省科技厅科技发展计划项目(201101004
20090246)
吉林省教育厅基金项目(吉教科合字(2009)第32号
吉教科合字(2010)第21号)
关键词
乳酸菌
诱导表达载体
NISIN
lactic acid bacteria
controlled expression vector
Nisin
