摘要目的 构建大鼠CD 40基因RNAi慢病毒载体.方法 针对大鼠CD40基因RNAi设计有效靶序列,合成靶序列的寡核苷酸序列.退火形成双链DNA,与经HpaI和XhoI双酶切后的GV118载体连接产生短发卡RNA慢病毒载体,PCR筛选阳性克隆,测序鉴定.随后进行Western blot外源筛选最佳干扰靶点、慢病毒包装及病毒滴度测定.结果 CD40siRNA成功插入慢病毒载体.慢病毒载体经293T细胞包装成功,测定病毒的滴度为2.0×1012 TU/L.结论 成功构建大鼠CD40基因的RNAi慢病毒载体,为后期进一步研究病毒性心肌炎发病机制及新的治疗方法提供工作基础.

abstractsObjective To construct the RNA interference (RNAi) vector targeting the CD40 gene in rats.Methods Effective target sequences that target at CD40 gene were designed,then the oligonucleotide sequences after annealing of the complementary strands were synthetized,the DNA fragments were connected to the GV118 vectors by double digestion with HpaI and XhoI,and the lentiviral vectors which expressed short hairpin RNA were constructed.The lentiviral vectors were identified by PCR and DNA sequencing.Western blot was employed to sort the best interfering targets exogenously,and lentiviral packaging as well as assaying viral titer were also accomplished.Results CD40 siRNA was successfully inserted into the lentiviral vectors and the lentiviral vector was packaged into 293T cells.The determination of virus titer was 2.0 × 1012 TU/L.Conclusions The RNA interference lentiviral vector targeting the CD40 gene in rats was constructed successfully,which will provide the foundation for further exploring the pathogenesis of viral myocarditis and novel therapies in future.