日本血吸虫中国大陆株肝期童虫cDNA文库的构建及鉴定

Construction and identification of schistosomulum cDNA library

目的 构建日本血吸虫肝期童虫的cDNA文库 ,以从中寻找新的抗病疫苗和诊断分子。方法 阳性钉螺逸出尾蚴 ,人工感染家兔 ,15d后剖杀家兔 ,门静脉灌洗收集童虫。提取其总RNA ,并反转录PCR合成cDNA ,将cDNA片段定向重组入噬菌体载体 ,包装后构建成cDNA文库。结果 反转录PCR产生的cDNA经电泳 ,分子量大小位于 4 0 0～ 35 0 0bp之间 ,经含有IPTG和x -Gal颜色选择平皿测定 ,初步提示重组效率为 90 %以上。随机挑取 6个重组克隆经自身环化成质粒后双酶切鉴定提示插入片段 4 0 0～ 12 0 0 pb不等。

Aim A liver stage schistosomulum cDNA library is costructed in order to look for new vaccine molecular Methods Rabbits were infected experimentally by cercariae released from positive snails and were sacrificed fifteen days later Schistosomulum were collected by douching the portal vein Total RNA was extracted from schistosomula and cDNA was synthesised by RT PCR The cDNA was recombinated oriently in to phage vector A schistosomula cDNA library was constructed after packaging Results The cDNA molecule weight range from 400～3 500bp was identified with agar electrophoresis The recombination rate were 90% measured by plate with IPTG and x Gal Six recombinated clones were pick out randomly and were self cycled into plasmid The plasmid were digested by two enzymes,the inserted fragments range from 400～1200bp Conclusion A liver stage schistosomula cDNA library has been successfully constructed for the first time