口蹄疫病毒VP1基因siRNA重组腺病毒的构建及其介导的RNAi抑制口蹄疫病毒复制的研究  

Construction of a recombinant adenovirus containing the VP1 gene siRNA of foot-and-mouth disease virus and adenovirus-mediated RNA interference against FMDV replication

作　　者：贾俊涛[1] 陈立军[1] 赵明秋[1] 琚春梅[1] 吕英然[1] 陈金顶[1] 

机构地区：[1]华南农业大学兽医学院,广东广州510642

出　　处：《中国兽医杂志》2010年第1期11-13,共3页Chinese Journal of Veterinary Medicine

基　　金：教育部"长江学者和创新团队发展计划"创新团队项目(TRT0723);广东省自然科学基金创新团队项目(5200638);广州市科技计划项目(2008Z1-E011);广东省自然科学基金项目(02099506025827)

摘　　要：本研究选择O型FMDV VP1基因上保守序列作为可能的干扰位点,合成了siRNA,将siRNA克隆到pSIREN-Shuttle中,获得siRNA重组表达载体pShuttle-VP1。用限制性内切酶PI-Sce I和I-Ceu I双酶切siRNA重组表达载体和腺病毒质粒载体pAdeno-X,利用体外连接法获得了重组质粒。经过PCR扩增、酶切鉴定及序列测定,证明成功构建了重组腺病毒质粒pAdeno-VP1。利用脂质体转染法,将Pac I线性化的重组腺病毒质粒pAdeno-VP1转染HEK293细胞,包装成重组腺病毒rAdeno-VP1。经过PCR鉴定正确后大量增殖,增殖的重组腺病毒经过浓缩、纯化得到了高滴度的病毒。用终点稀释法测定病毒滴度为2.5×109PFU/mL。将rAdeno-VP1感染IBRS-2细胞后,12 h后再加入100TCID50的FMDV O/HKN/2003。结果显示,重组腺病毒能够抑制FMDV的复制。In this study, the conservative sequence in VP1 gene of O type FMDV was chosen as possible interference site. A siRNA was synthesized. The siRNA was cloned into the plasmid pSIREN-Shuttle, then the siRNA recombined expression vector was obtained including pShuttle-VP1. The vector and the adenovirus vector pAdeno-X were digested with restriction endonuclease PI-Sce I and I-Ceu I and connected in vitro, then recombinant plasmid was obtained. The result of PCR, enzyme-digestion and sequencing suggested that the recombined adenovirus plasmid pAdeno-VP1 was constructed successfully. The recom- binant adenovirus plasmid pAdeno-VP1 was linearized with Pac I and then transfected into HEK293 cells mediated with liposomes to generate recombinant adenovirus rAdeno-VP1. After identified by PCR, high- titer virus was yielded by multiplicating, concentrating and purifying. The virus titer was 2.5 × 10^9 PFU/ mL determined with serial-dilution. The IBRS 2 ceils was infected with the recombined adenovirus rAdeno- VP1. The 100TCID50 of FMDV O/HKN/2003 was inoculated into the 1BRS-2 cells twelve hours later. The results showed that the recombined adenovirus inhibited FMDV replication.

关 键 词：口蹄疫病毒 RNA干扰 重组腺病毒 

分 类 号：S852.659.6[农业科学—基础兽医学]