摘要
利用pGPB14为启动子-信号肽序列探测载体,在大肠杆菌中克隆乳酸乳球菌总DNA中具有启动子-信号肽功能的DNA片段。共得到42个具有红霉索、氨苄青霉素双重抗性的转化子。转化子的氨苄青霉素抗性水平在100～800μg/ml之间,β-内酰胺酶活力大多积累于周质空间,说明重组质粒中的外源插入片段确实具有发动转录和促进分泌的功能。Southern杂交结果表明,插入片段的确来源于乳酸乳球菌总DNA,其大小在80～400bp之间。DNA序列测定发现pSEQ8和pSEQ12中插入片段分属于pSEQ4和pSEQ17插入片段的一部分。在测序的4个DNA序列中都找到了启动子和起始密码子。其中2个含有典型的S.D.序列和非典型的信号肽序列,其他2个则没有发现典型的S.D.序列和信号肽序列。另外发现启动子上游序列对启动子转录起始的效率具有促进作用。
Promoter and signal peptide function fragments from Lactococcus lactis were clonedin E. coli using a promoter-signal sequence probe vector pGPB14. Forty-two cloneswere obtained, whose level of resistance to ampicillin ranged from 100 to 8000 μg/ml.Eight clones were selected for β-lactamase distribution assay. The β-lactamase activitywas found mainly in the periplasm, which indicated successful secretion of the enzyme.Southern hybridization test demonstrated that the inserted fragments were indeed from L.lactis. Restriction enzyme analysis revealed that the size of inserted fragments rangedfrom 80bp to 400 bp, four of which were sequenced on the vector pGEM-3Zf. Itwas found that the inserted fragments of pSEQ8 and pSEQ12 turned out to be part ofthe inserted fragments of pSEQ4 and pSEQ17 respectively. Promoter and translationinitiation codon were found among all four fragments signal sequenced. One typical S.D. sequence and one atypical signal sequence were found in pHSB4 and pHSB8,while the other two contained no typical S. D. sequence and signal peptide sequence.In addition, it was found that the upstream regions of the promoter contributed to theefficiency of transcription initiation.
基金
中国科学院"八五"重点科研项目
关键词
乳酸乳球菌
启动子
信号肽
大肠杆菌
基因克隆
L.lactis
promoter-signal sequence
E.coli
Gene cloning
DNA sequencing
