载体表达的siRNA分子对猪圆环病毒2型复制的抑制作用

Inhibition of porcine circovirus type 2 replication by the plasmid vector expressing siRNAs

【目的】寻找一种基于RNA干扰技术的猪圆环病毒2型感染的防控方法。【方法】根据猪圆环病毒2型毒株基因组核苷酸序列,设计了3条特异性小干扰RNA(short interfering RNA,siRNA)分子,其中2条针对猪圆环病毒1型和2型复制酶基因(rep),1条针对猪圆环病毒2型核衣壳蛋白基因(cap),将合成的DNA片段退火形成双链,分别连接到RNAi-Ready pSIREN-RetroQ ZsGreen载体鼠源U6启动子下游,转化大肠杆菌得到阳性克隆,测序鉴定后分别命名为Retro-SH1,Retro-SH4,Retro-SH6。用上述质粒转染PCV2感染前、后的Dulac细胞及肌肉注射PCV2感染前、后的BALB/c小鼠,应用实时定量PCR试验评价其对病毒在细胞及小鼠体内复制的抑制作用,免疫组化法检测脾脏中病毒的存在。【结果】感染PCV2前或后转染500ng Retro-SH1,Retro-SH4,Retro-SH6质粒能有效抑制PCV2在Dulac细胞上的复制,抑制率最高可达99%以上,对10株不同来源的临床分离株在细胞中复制的抑制作用同样明显,且不同毒株间差异不大。动物试验中,肌肉注射10μg上述不同siRNA分子对小鼠体内PCV2的复制有一定的抑制作用,其抑制率在26%至99%之间。【结论】载体表达的siRNA分子可能成为防控猪圆环病毒2型感染的一种新工具。

[Objective] To develop siRNA as a potential tool for control of porcine circovirus 2 （PCV2） infection. [Methods] 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 DH5α 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. [Results] 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%. [Conclusion] The siRNAs specific to PCV2 expressed by vectors should be potential in the control of the diseases related to PCV2 infection.