伪旋毛虫肌幼虫cDNA表达文库的构建及筛选

Construction and screening of the cDNA expression library for muscle larvae of Trichinella pseudospiralis

目的获取伪旋毛虫(Trichinella pseudospiralis)肌幼虫时期抗原性基因。方法利用λZAP载体构建伪旋毛虫肌幼虫cDNA表达文库;以感染伪旋毛虫60天后的猪血清为抗体探针,对伪旋毛虫肌幼虫cDNA文库进行免疫学筛选,对筛选出的强反应原性克隆进行测序,利用相关分子生物学软件进行序列分析。结果构建的伪旋毛虫cDNA表达文库的库容量为0.55×106pfu,重组率为95.6%,扩增后文库滴度为1.2×1010pfu/mL。从2.0×105个重组噬菌体筛选获得阳性克隆27个。序列分析结果表明这27个阳性克隆共编码7种cDNA分子,其编码的类似蛋白分别为伪旋毛虫Tp21kDa蛋白、伪旋毛虫Tp28kDa蛋白、蛋白酶体激活因子亚单位(Proteasome activator subunit)类蛋白、Nudix水解酶(Nudix hydrolyase)类蛋白、凝集因子(Condensin)类蛋白、尿嘧啶糖基化酶(uracil glycosylase)类蛋白和一个未知蛋白。结论获得两个反应原性较强且高拷贝的抗原基因,其分别编码Tp21kDa蛋白及蛋白酶体激活因子亚单位。

The cDNA expression library for muscle larvae of Trichinella pseudospiralis was constructed with lambda ZAP vector and screened using the swine serum from T. pseudospiralis 60 days post infection（60 dpi） as the antibody probe. Meanwhile, the positive clones screened were sequenced and the sequences were analyzed by means of the related softwares of bioinformatics. It was found that the size of the primary library was 0.55 × 10^6 pfu with a recombination rate of 95.6% and the titer of the amplified library was 1.2 × 10^10. From 2 × 10^5 recombinant clones of the cDNA library, 27 positive clones were obtained and these clones encoded 7 unique cDNA molecules coding for Tp21kDa and Tp28kDa proteins of T. pseudospiralis, proteasome activator subunit, nudix hydrogenase- like protein, condensing-like protein, uracil glycosylase-like protein and an unknown protein.