摘要
目的克隆和表达日本血吸虫(Sj)腺苷脱氨酶(ADA)编码基因,分析该基因的系统发育及预测其编码蛋白的空间结构。方法根据表达序列标签(EST)测序的结果设计引物,PCR法从含有SjADA基因的cDNA克隆中扩增得到该编码基因片段,亚克隆入原核表达载体pET32中表达,表达的融合蛋白用螯合琼脂糖凝胶FF亲和层析纯化。采用MrBayes算法构建系统发育进化树,用自动蛋白注释工具(DS GeneAtlas)软件模拟SjADA的蛋白空间结构。结果获得SjADA基因全长为1 059 bp的编码序列,并克隆、表达和纯化了重组蛋白(SjADA)。生物信息学分析显示,该基因与曼氏血吸虫的同源基因之间的序列一致性仅25%,属于不同的亚家族。其空间结构与PDB模板1A4M的一致性为41%,具有相似的二级结构和相应的活性位点残基。结论获得了SjADA重组蛋白。提示日本血吸虫嘌呤补救合成代谢途径与曼氏血吸虫有差异。
Objective To clone and express a new protein adenosine deaminase of Schistosoma japonicum （SjADA）. Method Specific primers were designed according to the EST sequence and used for amplification of the encoding sequence from the eDNA clone containing SjADA. The gene was sub-cloned into pET32 plasmid and expressed in E.coli BL31 （DE） pLys strain and the recombinant proteins were purified by chelating sepharose FF. The structure and phylifunetions of this protein were analyzed by MrBayes and GeneAtlas. Result The full length sequence of SjADA gene was obtained and the recombinant protein was cloned, expressed and purified successfully. The bioinformatics analysis showed that the identity of SjADA and SmADA gene sequence was only 25% and they belonged to different subfamily. The structure of SjADA had 41% identity with it＇s PDB template 1A4M. Conclusion The recombinant protein of SjADA has been obtained. The purine salvage pathway of S. japonicum may be different with that of S. mansoni.
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2007年第1期6-11,共6页
Chinese Journal of Parasitology and Parasitic Diseases
基金
国家自然科学基金(No.30570429)
上海市科委"启明星"人才计划项目(No.04QMX1455)~~
