摘要
在完成小花棘豆毒素 95 %氨基酸序列的基础上 ,根椐已知的氨基酸序列 ,设计合成了特异简并引物 .以小花棘豆总RNA为模板 ,逆转录合成cDNA第一链 ,用置换法合成双链cDNA .用特异引物对此双链cDNA进行PCR扩增 ,将扩增后的目的基因与用SmaⅠ酶切的质粒pUC 18连接 ,转化大肠杆菌JM10 7.筛选阳性克隆进行序列分析 ,获得了OXY基因的全部序列 .经测序后测得基因序列与原氨基酸序列对照完全一致 .GenBnak数据检索说明 ,OXY基因编码序列确定是一个从未报道的序列 .此研究结果对该毒素的应用研究奠定了基础 .
Based on completing 95% amino acid sequence of Oxytropis glabra DC hemolyti toxicn (OXY), OXY's special primer was synthesized according to OXY's amino acid sequences. The first strand cDNA was obtained from reverse transcription of total RNA of Oxytropis glabra DC. The second strand cDNA was synthesized by method of displacement. DNA fragment was amplified by PCR with this cDNA as template. This DNA fragment was insert into pUC18 at Sma Ⅰ site. The recombinant plasmid was used to transform the competent E.coli JM107 cell. A positive clone was identified by restriction analysis. DNA sequence analysis indicated that the cloned 420 bp DNA fragment carried the entire coding sequence and coincided with OXY amino acid sequences. OXY gene has been obtained. GenBank search showed that the sequence of OXY had never been reported.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2005年第2期176-179,共4页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金 (No .3 9860 0 2 5 )
国家"八六三"计划项目 (No .HB0 2 0 10 1)资助~~
