摘要
构建乳酸乳球菌同源整合载体,实现外源基因在乳酸菌中的同源重组和转入。PCR扩增红霉素抗性基因,连接到T载体中,构建载体pMDE;根据乳酸乳球菌Lactococcus lactis1.2472全基因组中usp基因序列设计引物,PCR扩增usp基因两端的同源重组臂LB和RB基因序列,PCR产物经回收后,克隆至pMDE载体中,获得同源重组载体pMDELR,电转化至Lactococcus lactis1.2472感受态细胞中,挑选阳性克隆,采用PCR和SDS-PAGE方法进行鉴定。结果表明:成功扩增同源重组臂LB和RB,红霉素抗性基因替换了乳球菌中的usp基因,整合到了乳球菌基因组中。
The purpose of this study was to construct homologous integration vector of L.lactis and to implement the ectogenic genome integration into L.lactis genome successfully.The Emr gene was amplified by PCR and cloned into T vector,then the pMDE vector was obtained.Using chromosome DNA of L.lactis 1.2472 as template,the left and right arm of usp were amplified by PCR and purified by purification kit.The PCR product was cloned into pMDE vector,then the homologous recombination pMDELR vector was obtained,and transformed into L.lactis 1.2472 competent cells by electricity.The positive clones were selected,PCR and SDS-PAGE were employed to conform it.Homologous recombination arms LB and RB were amplified successfully,the usp gene was replaced by erythromycin resistance gene,and was integrated into the genome.The method laid foundations for the construction of new expression vector of L.lactis and the expression of foreign gene in lacticacid bacteria.
出处
《吉林农业大学学报》
CAS
CSCD
北大核心
2012年第6期628-633,共6页
Journal of Jilin Agricultural University
基金
国家自然科学基金项目(81001342)
军内“十一五”科技攻关项目(06G127)
吉林省高新技术产业发展项目(2010)
长春市科技特派员行动计划项目
关键词
乳酸乳球菌
usp基因
同源重组
表达载体
Lactococcus lactis
usp gene
homologous recombination
expression vector
