大鼠CD40基因RNAi慢病毒载体的构建与鉴定  

Construction and identification of RNA interference lentiviral vector targeting CD40 gene in rats

作　　者：王介忠[1] 韩波[1] 高聆[2] 杨文巍[1] 

机构地区：[1]山东大学附属省立医院小儿心脏科,济南250021 [2]山东大学附属省立医院中心实验室,济南250021

出　　处：《中华实用儿科临床杂志》2013年第10期745-748,共4页Chinese Journal of Applied Clinical Pediatrics

基　　金：山东省自然科学基金（ZR2011HM029）

摘　　要：目的构建大鼠CD。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Hpal和Xhol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Westernblot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2．0×10^12TU／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。Objective 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 oligonncleotide 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 interfe- ring targets exogenously,and lentiviral packaging as well as assaying viral titer were also accomplished. Results CD4o siRNA was successfully inserted into the lentiviral vectors and the lentiviral vector was packaged into 293T cells. The determination of virus titer was 2.0×10^12TU/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.

关 键 词：CD40 RNAI 慢病毒载体 病毒性心肌炎 

分 类 号：R54[医药卫生—心血管疾病]