摘要
目的通过天然分子疫苗免疫猪血清作为探针筛选日本血吸虫尾蚴cDNA文库,应用表达序列标签获得日本血吸虫新基因。方法制备天然分子免疫猪血清,将筛选的尾蚴cDNA文库中大肠杆菌XL1-Blue侵入大肠杆菌BM25.8后环化成质粒,筛选出已转入质粒的菌株。提取质粒DNA,进行测序,得到一个表达序列标签(EST)。通过NCBI网站的BLASTx及BLASTn公共数据库,编辑分析EST序列并进行同源性比较。结果从26个克隆样品中筛选出8个阳性克隆,其中有一个新基因(GenBank登录号DQ097517),该基因全长861bp,有完整的阅读框架,与蜜蜂(Apismellifera)SMC3蛋白编码的mRNA基因序列同源性最高。结论疫苗免疫猪血清可作为筛选日本血吸虫新基因的有效探针,新发现1个日本血吸虫相关基因。
Objective To discover the novel genes in Schistosoma japonicurn cereafia by expressed sequence tag （EST）. Methods Sera prepared from pig vaccinated with the natural molecular vaccine were used to screen for S.japonicum cercaria eDNA libray. The E.coli XLl-blue in Sj cercariae eDNA library was used in the transformation of E.coli BM25.8.E.coli clones containing the plasmids were cultured in LB medium supplemented with aminobenzylpenicillin. Colonies were then selected for plasmid extraction.After DNA extraction, the plasmids were sequenced and then analyzed with a sequencing primer to obtain the EST. After sequence editing of the resulted EST,comparison of these sequences with those listed in the GenBank database was performed for identity analysis.The new sequences generated from the analysis were submitted to GenBank and the accession numbers were obtained.Results Eight positive clones were screened from 26 isolated colonies. One of the clones similar with the SMC3 protein of Apis mellifera was registered on Genbank. Conclusion The immune sera from pig vaccinated with the natural molecular vaccine can be used as a probe to screen for new genes in the Si eercaria eDNA.
出处
《热带医学杂志》
CAS
2006年第3期260-262,259,共4页
Journal of Tropical Medicine
基金
国家"十.五"重大科技专项资助项目(No.2002AA2Z3343)
国家"863"血防重大专项(No.2004AA2Z3530)
教育部"985行动计划"(No.03-985-3)
湖南省"十五"重点学科建设专项经费(No.2003-985-3-7)。
关键词
日本血吸虫
天然分子疫苗
表达序列标签
序列分析
Schistosomajaponicum
natural molecular vaccine
expressed sequence tag
sequence analysis
