华支睾吸虫cDNA表达文库的构建及初步筛选

Construction and identification of a cDNA expression library from Clonorchis sinensis

目的 构建华支睾吸虫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 提取华支睾吸虫总RNA ;用Clontech公司SMARTTMcDNA文库构建试剂盒操作方法 ,进行反转录合成双链cDNA ;PCR产物纯化后 ,进行SfiI酶切 ;用ChromaSpin 40 0柱将酶切产物进行分级分离 ,回收 0 .4～ 4kb的组分 ,并与λTriplEx2载体连接、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及鉴定。 结果 未扩增文库滴度达 7.5× 10 7pfu/ml ,扩增文库滴度达 2 .7× 10 8pfu/ml ;用载体两端的引物进行PCR鉴定 ,显示 :所选噬菌体中均含有重组的cDNA ,大小在 5 0 0bp以上。 结论 已成功地获得一高质量的华支睾吸虫cD

Objective To screen specific-antigen gene for diagnosis and antigen gene for candidate vaccine,a cDNA expression library of Clonochis sinensis(Cs) was constructed. Methods Total RNA from Cs was extracted and cDNA was synthesized by SMART TM cDNA library protocol.Products were digested by SfiⅠ,and fractionated with CHROMA SPINT 400 column. The cDNA with size of 0.4～4kb were recycled and ligated to λTriplEx2 Vector. The λphage packaging reaction for the ligations was performed to produce a unamplified library. After the unamplified library and the percentage of its recombinant clones was respectively tittered. The library amplification and the titer of the amplified library were executed, successively .Ten phages were randomly selected and amplified using primers from vector by PCR to test the quality of obtained library. Results The titers of unamplified and the amplified library are 7.5×10 7pfu/ml and 2.7×10 9 pfu/ml respectively.The percentage of recombinant was nearly 100% . Result of PCR indicated that all the selected phages possessed the recombinant cDNA , and that these cDNA are beyond 500bp. Conclusion A qualified cDNA library from Cs is constructed successfully .