华支睾吸虫后囊蚴cDNA表达文库的构建及鉴定

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

构建华支睾吸虫后囊蚴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,也为进一步研究华支睾吸虫后囊蚴感染宿主的信号传导机制、探究其致病机理提供新的起点。从中国东北疫区(镇赉县)麦穗鱼肉里分离收集华支睾吸虫囊蚴,采用Trizol Reagent提取其总RNA,Oligo(dT)纤维素柱纯化mRNA,反转录合成第1链cDNA及第2链cDNA,加EcoRⅠ接头,经XhoⅠ酶切,用CHROMA SPIN-400柱离心层析纯化后,与载体λZAP Express连接,Gigapack Ⅲ Gold包装蛋白体外包装,构建华支睾吸虫后囊蚴cDNA表达文库。结果表明,构建的华支睾囊蚴cDNA表达文库的库容量为9.15×105PFU,重组率为99.5%,扩增后文库滴度为1.6×1010PFU/mL。成功构建了一高质量的华支睾吸虫后囊蚴cDNA表达文库。

The cDNA expression library of Clonochis sinensis metacercaria was constructed for selecting stage specific diagnosis antigen gene or vaccine candidate gene.Total RNA of leucocytes was then extracted,pooled together and mRNA was isolated with a column of oligo（dT） cellulose.And cDNA was synthesized by Stratagene cDNA library protocol.The first and second strand of cDNA were synthesized using Stratagene Uni-ZAP XR cDNA synthesis kit,then ligated to EcoR I adapters and digested with Xho Ⅰ.After size fractionated with CHROMA Spin-400 column cDNA were ligated into Uni-ZAP vector and packaged in vitro to construct cDNA libraries.It was found that the size of the primary library was 9.15×10^5 PFU with a recombination rate of 99.5% and the titer of the amplified library was 1.6×10^10 PFU/mL.cDNA library for Clonochis sinensis Metacercaria has been constructed.