我国旋毛虫Ts254 DNA探针制备及序列测定

PREPARATION AND SEQUENCE ANALYSIS OF A Ts254 DNA PROBE FROM A TRICHINELLA SPIRALIS ISOLATE IN CHINA

我国云南猪体旋毛虫分离株（ＢＳ）经随意扩增的多态ＤＮＡ（ＲＡＰＤ）方法产生的２５４ｂｐ条带，以地高辛（ｄｉｇｏｘ－ｉｎｉｎ）标记制成探针（Ｔｓ２５４），将此探针与７株旋毛虫总ＤＮＡ进行斑点杂交，出现明显的杂交斑点，检出的ＤＮＡ量为１ｎｇ。但与其它相关寄生虫、质粒ｐＢＲ３２２、昆明株小鼠肌肉ＤＮＡ均未出现杂交反应，表明Ｔｓ２５４探针具有一定的特异性和较高的敏感性。将此Ｔｓ２５４ＤＮＡ片段克隆进Ｍ１３ｍｐ１８／１９噬菌体，扩增后，筛选阳性重组子，抽提单链后测序。经Ｇｅｎ－Ｂａｎｋ序列数据库检索，Ｔｓ２５４序列与真核ＤＮＡ及结构ＲＮＡ序列无有意义的同源性，此旋毛虫ＤＮＡ序列为我国首次报道。

A 254bp DNA band from BS isolate of Trichinella spiralis by RAPD was isolated and labelled with digoxinin as Ts254 DNA probe.The sensitivity of the probe was demonstrated in the dot blot hybridization to detect 1ng genomic DNA.The Ts254 DNA probe reacted with all the 7 isolates of Trichinella spiralis ,with the DNA from other related parasites,plasmid pBR322 and muscle tissue of mouse.The 254bp DNA band was cloned into M13mp18/19 vectors and the insert was sequenced from both directions.Comparison of this 254bp DNA with the GenBank sequence data showed no significant homologies to eukaryotic DNA or structural RNA sequences.This sequence of Trichinella spiralis has not been reported from China.