华支睾吸虫囊蚴cDNA表达文库的构建及鉴定

Construction and Identification of a cDNA Expression Library from Clonorchis sinensis Metacercaria

目的 构建华支睾吸虫囊蚴cDNA表达文库,为筛选特异诊断抗原和疫苗候选抗原奠定基础。方法提取华支睾吸虫囊蚴总RNA;用Clontech公司SMARTTM cDNA文库构建试剂盒并按其操作方法进行,进行反转录合成双链cDNA。PCR产物经蛋白酶K消化、纯化后,进行SfiI酶切;用ChromaSpin 400柱将酶切产物进行分级分离,经1.1％琼脂糖凝胶电泳鉴定,回收0.4～4kb的组分,并与λTriplEx2载体连接;连接产物经体外蛋白包装,产生未扩增文库;检测未扩增文库滴度和重组效率后,进行文库的扩增,并测定扩增文库的滴度;随机挑取9个噬菌斑,用载体克隆位点两端的引物进行PCR扩增,以检测所构建的cDNA文库的质量。结果 未扩增文库滴度达7.0×107pfu/ml,扩增文库滴度达2.5×108pfu/ml;用载体两端的引物进行PCR鉴定,结果显示:扩增文库中,含1kb以上的占30％左右,1kb～500bp占69％。结论已成功地获得一高质量的华支睾吸虫囊蚴cDNA表达文库。

Objective To select stage specific-antigen gene for diagnosis and antigen gene for vaccine candidate, a cDNA expression library of Clonochis sinensis metacercaria (Csm) was constructed. Methods Total UNA from C. sinensis metacercaria was extracted and cDNA was synthesized by SMART?cDNA library protocol. Products were digested by proteinase K and Sfi I , and fractionated with CHROMA SPINT 400 column. The fractions were characterized by 1.1% agarose/EB gel electrophorese. The cDNA with size of 0.4 - 4kb were reamplified and h'gated to λ TriplEx2 vector. The Xphage packaging reaction for the ligated DNA was performed to produce an unamplified library. Then, the phage titer the percentage of the recombinants of the unamplified library were estimated. Ten phages were randomly selected and amplified using primers from vector by PCR to test the quality of obtained library. Results The tilers of unamplified and the amplified library were 7.0 × 107pfu/ml and 2.5 × 108 pfu/ml respectively. 30% of the recombinant contained an insert beyond 1kb, while the insert of 69% of the recombinants was between 500bp - 1000bp. Condution A high quality cDNA library from C. sinensis was constructed successfully.