摘要
目的 识别和克隆日本血吸虫新基因。方法 对本实验室获得的EST进行同源性分析 ,识别血吸虫新基因 ;根据EST设计引物 ,用锚着PCR法从cDNA文库中扩增出新基因全长cDNA ,并将其克隆入原核表达载体 pGEX - 4T - 1,用生物信息学技术对获得的编码基因进行结构与功能的分析。结果 EST同源性分析得到一个完整编码基因 ,开放阅读框(ORF) 4 95bp ,编码 16 4个氨基酸 ;锚着PCR法获得另一EST的 3’端所缺序列 ,得到完整编码阅读框 ,其ORF 999bp ,编码 332个氨基酸。利用生物信息学技术确定它们分别为日本血吸虫亲环素A(CyPA)和乳酸脱氢酶 (LDH)的编码基因。重组质粒经双酶切及测序鉴定证明日本血吸虫CyPA和LDH原核表达质粒构建成功。 结论 克隆到日本血吸虫亲环素A和乳酸脱氢酶编码基因的全长cDNA 。
Aim To recognize and clone the novel genes of Schistosoma japonicum(Sj) Methods The ESTs obtained in our laboratory were analysed by homologous searching,the novel genes were recognized According to the interested EST,the full length cDNA of the novel gene was obtained by anchored PCR and directed sequencing of novel clone from cDNA library The cDNA sequence were cloned into pGEX 4T 1 vector and analysed by bioinformatics Results A new cDNA sequence was obtained by homologous searching,the sequence contains a full length sequence with 495bp ORF,which encodes 164 amino acid residues Another full length cDNA was obtained by anchored PCR which has a 999bp ORF encoding 332 amino acid residues The two sequences were cloned into pGEX 4T 1 vector and identified by restriction analysis and sequencing The former gene was identified to be the gene encoding cyclophilin A and the other was the gene encoding lactate dehydrogenase Conclusion The full length cDNA sequences encoding Sj cyclophilin A and lactate dehydrogenase have been firstly sequenced and cloned,which give the basis for further study
出处
《中国人兽共患病杂志》
CSCD
北大核心
2003年第3期37-40,共4页
Chinese Journal of Zoonoses
基金
国家自然科学基金课题 (3 0 0 70 683 )
国家教育部博士点基金课题(2 0 0 0 45 )
广东"2 11工程"重点建设项目 (98169)资助
广东省首批自然科学团队研
关键词
日本血吸虫
亲环素A
乳酸脱氢酶
编码基因
克隆
序列分析
Schistosoma japonicum
Chinese strain
Cyclophilin A
Lactate dehydrogenase
clone
sequence analysis
