Wang Haiyan, Liu Wenbo, Gao Song, Liu Xiufan
Key Laboratory of Animal Infectious Diseases of Ministry of Agriculture, Yangzhou University, Yangzhou 225009, China.
Wei Sheng Wu Xue Bao. 2008 Nov;48(11):1507-13.
To develop siRNA as a potential tool for control of porcine circovirus 2 (PCV2) infection.
We designed two short interfering RNAs (siRNAs) related to the replicase (rep) gene of porcine circovirus and one related to capsid (cap) gene of PCV2 basd on the genomic sequences of the viruses deposited in GenBank. The corresponding DNA fragments were synthesized, annealed and ligated into the downstream of the mouse originated U6 promoter of the RNAi-Ready pSIREN-RetroQ ZsGreen vector. As controls, the siRNAs specific to Luciferase contained in the vector kit and negative fragments with no similarities to any known species were also included. We transformed the recombinant plasmids into the host bacterium DH5a and positive clones were selected. The positive clones were sequenced and five clones carried the correct inserts. They were designated as Retro-SH1, Retro-SH4, Retro-SH6,Retro-Luc and Retro-NC. To test whether the siRNAs specific to PCV expressed by the vector could inhibit the virus replication, we evaluated the inhibition effect on PCV2 replication both in Dulac cells and experimental mice using real-time PCR and immunohistochemistry.
The results showed that the purified plasmids could strongly inhibit the replication of the PCV2 virus and the inhibition rate reached to 99% in cell culture. In the animal experiments, siRNAs expressed by the plasmids could inhibit the replication of the PCV2 virus and the inhibition rate varied from 26% to 99%.
The siRNAs specific to PCV2 expressed by vectors should be potential in the control of the diseases related to PCV2 infection.
开发小干扰RNA（siRNA）作为控制猪圆环病毒2型（PCV2）感染的潜在工具。
根据GenBank中已存病毒的基因组序列，我们设计了两条与猪圆环病毒复制酶（rep）基因相关的小干扰RNA（siRNA）以及一条与PCV2衣壳（cap）基因相关的小干扰RNA。合成相应的DNA片段，退火后连接到RNAi-Ready pSIREN-RetroQ ZsGreen载体的小鼠源U6启动子下游。作为对照，还包括载体试剂盒中含有的针对荧光素酶的特异性siRNA以及与任何已知物种均无相似性的阴性片段。我们将重组质粒转化到宿主菌DH5α中，并筛选出阳性克隆。对阳性克隆进行测序，有五个克隆携带正确的插入片段。它们被命名为Retro-SH1、Retro-SH4、Retro-SH6、Retro-Luc和Retro-NC。为了检测载体表达的针对PCV的siRNA是否能抑制病毒复制，我们使用实时荧光定量PCR和免疫组化评估了其对杜拉克细胞和实验小鼠中PCV2复制的抑制作用。
结果表明，纯化后的质粒能强烈抑制PCV2病毒的复制，在细胞培养中的抑制率达到99%。在动物实验中，质粒表达的siRNA能抑制PCV2病毒的复制，抑制率在26%至99%之间。
载体表达的针对PCV2的siRNA在控制与PCV2感染相关的疾病方面应具有潜力。
