摘要
以大肠杆菌pSLM004菌株为始发菌株,利用合成的一对与ST_1基因特定序列相匹配的引物,通过PCR扩增出了ST_(1b)基因。该基因片段扩增物经适当纯化,与克隆载体pUC18的HincⅡ切点平端连接,转化到受体菌JM101中。利用Ap抗性作为压力选择标志以及LacZ基因插入失活显色反应,在Ap/Xgal-IPTG培养基上,筛选出转化子。将白色菌落转化子经酚/氯仿法抽提重组质粒,利用所扩增ST_(1b)基因片段上所设计的BglⅡ位点及pUC18载体具有的PvuⅡ切点,经酶切反应鉴定重组子,得到了理想重组子pBST2-6。该重组质粒经双脱氧法双向核苷酸序列分析,确定了插入的ST_(1b)基因与载体的连接向位及其全部核苷酸序列。
A gene encoding a heat-stable enterotoxin (ST1b) was amplified by PCR from an E. coli strain pSLM004. The gene fragments amplified were purified and ligat-ed to Hinc I -cleaved plasmid PUC18. The ligation mixture was used to transform E. coli JM101. The transformants were screened on ampicillin/Xgal-IPTG plates. One re-combinant, pBST2-6, was identified with restriction endonucleases (Bgl II and Pvu II). The nucleotide sequence analysis of ST1bgene reveals the DNA sequence of plasmid pB-ST2-6. The present study on subclone and DNA sequence of ST1bgene will provide a valuable information for the further investigation of the enterotoxin gene.
出处
《中国兽医学报》
CAS
CSCD
1994年第3期232-235,共4页
Chinese Journal of Veterinary Science
基金
军队医药卫生科研基金资助项目
