日本血吸虫新基因精氨酸酶的扩增及序列分析

Schistosoma japonicum:amplication and sequence analysis on new gene encoding arginase

目的 获取并真核克隆日本血吸虫新基因精氨酸酶全长cDNA。方法 对本实验室曾获得精氨酸酶基因利用生物信息学进行分析 ,发现其 5’端尚缺一段序列 ;根据已知序列设计引物 ,用 5’端单巢式PCR从尾蚴文库中扩增 ,以获得精氨酸酶基因完整的阅读框 (ORF) ;用生物信息学技术对获得的精氨酸酶编码基因进行结构与功能的分析 ;并将新基因的完整编码阅读框克隆入真核表达载体pCDNA3 1。结果 用 5’端单巢式PCR从尾蚴文库中获得了精氨酸酶 5’端所缺序列 ,得到其完整的ORF ,其ORF长 10 95bp ,编码 36 4个氨基酸。利用生物信息学技术鉴定其为日本血吸虫精氨酸酶的完整cDNA序列。重组质粒经双酶切DCR及测序鉴定证明日本血吸虫精氨酸酶真核表达质粒构建成功。结论 成功获得、识别、扩增并克隆日本血吸虫精氨酸酶编码基因的全长cDNA。

Objective To recognize and clone the novel gene named arginase of Schistosoma japonicmn (Sj).Methods The arginase gene obtained in our laboratory were analyzed by bioinformatics,the gene were recognized to be incomplete.The full-length cDNA of arginase gene was obtained by 5'-end nested PCR and was made sure by sequencing in Shenyou company.Specific primers were synthesized and were used to amplify arginase gene by PCR from cercariae cDNA library.Then,the cDNA sequences were cloned into pCDNA3.1 vector and analyzed by bioinformatics.Results A new cDNA sequence was obtained.The sequence contained a full-length sequence with 1?095?bp ORF,which encodeed 364 amino acid residues.The new gene was identified to be the gene encoding arginase.Its sequences were cloned into pCDNA3.1 vector and identified by restriction analysis,PCR amplication and sequencing.Conclusion The full-length cDNA sequences encoding Sj arginase were firstly sequenced and cloned,which gave the basis for further study.