猪带绦虫六钩蚴cDNA文库的构建与筛选  

Construction and Screening of a cDNA library of Taenia solium on cospheres

作　　者：唐雨德[1] 陆承平[2] 

机构地区：[1]南京军区军事医学研究所,江苏南京210002 [2]南京农业大学动物医学院,江苏南京210095

出　　处：《中国兽医学报》2003年第6期567-569,共3页Chinese Journal of Veterinary Science

摘　　要：在体外将猪带绦虫虫卵孵化为有活力的六钩蚴。采用商品化试剂盒抽提六钩蚴总 RNA、m RNA,反转录为双链c DNA,再加 Eco R Adapter。在去除小片段后 ,dsc DNA与 λgt11噬菌体 DNA连接 ,经蛋白包装 ,构建了猪带绦虫六钩蚴λgt11c DNA文库。该文库效价为 3.5× 10 9pfu/ m L ,重组 DNA片段大小为 0 .5～ 3.2 kb,平均为 1.6 kb,蓝白斑比 1∶ 9。用抗体探针对文库进行免疫学筛选 ,在消除非特异性反应的基础上筛选约 10 6 重组子 ,共得到 118株强阳性克隆 ;应用 PCR鉴定上述部分阳性克隆 ,均扩增到 0 .5 kb以上的片段。结果显示 ,构建的文库合格 ,含六钩蚴所有抗原基因 ,可用于六钩蚴 c DNA克隆的筛选 ;用免疫学与 PCR联合筛选 c DNA文库 。Taenia solium oncospheres were hatched and cultivated in vitro from the proglottid eggs.Total RNAs and mRNAs were extracted from the hatched oncospheres with commercial kits.Moreover,single strand cDNAs and double strand cDNAs were synthesized in turn,double strand cDNAs were ligated to the EcoRⅠ Adapter.After purified,the dscDNAs were ligated then with arms of the bacteriophage λgt11 and further packaged in vitro to set up the cDNA library.Titering and amplification of the library was carried out with the E.coli strain Y1090.The size of recombinant cDNAs was detected by PCR with the forward and reverse sequencing primers of λgt11 phage.The results revealed that the library contained 3.5×10 9 pfu/mL,the size of recombinant cDNA was 0.5 to 3.2 kb,and the ratio of blue and white phages was 1 to 9.The cDNA library was screened by immunological method.After eliminating the non specific reation,10 6 recombinants were screened with the blend sera of rabbit anti oncospheres and cysticerci and 118 strongly positive clones were obtained,some of the results were confirmed by PCR.The results revealed that the constructed cDNA library of Taenia solium oncosphere is a highly efficient one and lays solid foundation for screening and cloning specific genes.The combination with immunological method and PCR which was used to screen cDNA library could eliminate false positive.

关 键 词：猪带绦虫 六钩蚴 CDNA文库 囊虫病 免疫 抗原 筛选 

分 类 号：S858.28[农业科学—临床兽医学]