Jiang Zaixue, Wu Jiajun, Jiang Mei, Xie Yilun, Bu Wandi, Liu Canbin, Zhang Guihong, Luo Manlin
Key Laboratory of Zoonosis Prevention and Control of Guangdong Province, College of Veterinary Medicine, South China Agricultural University, Guangzhou, China.
Front Microbiol. 2020 Jun 9;11:1067. doi: 10.3389/fmicb.2020.01067. eCollection 2020.
Porcine circovirus type 3 (PCV3), which currently lacks effective preventive measures, has caused tremendous economic losses to the pig husbandry. Obtaining the strain of PCV3 is the key to preparing related vaccines and developing corresponding antiviral drugs. In this study, according to the linear sequence of PCV3 DNA published on GenBank, the sequence was rearranged with SnapGene gene-editing software, and after rearrangement, the dIII restriction endonuclease site was added to the end of the linear DNA, so that both ends have the same restriction endonuclease site. On this basis, the rearranged linear DNA is obtained by gene synthesis, PCR amplification, DNA purification, etc., and is digested and connected to obtain cyclized DNA. PCV3 infectious clones were obtained by transfecting 3D4/21 cell lines. The obtained PCV3 was identified by PCR, Western blotting, and indirect immunofluorescence tests. The results showed that this study successfully obtained the strain of PCV3 . To further evaluate the pathogenicity of the obtained PCV3 infectious clones, this study established an animal model of Kunming mice infected with PCV3. The results of RT-PCR, Western blotting and immunohistochemistry showed that PCV3 can infect myocardium and alveoli of Kunming mice, but no PCV3 was detected in other tissues. The above studies indicate that PCV3 circular DNA can be used to construct PCV3 infectious clones. This research will provide a new method for the construction of circular DNA viruses and lay the foundation for the research and pathogenesis of PCV3 vaccine.
猪圆环病毒3型(PCV3)目前缺乏有效的预防措施,给养猪业造成了巨大的经济损失。获得PCV3毒株是制备相关疫苗和研发相应抗病毒药物的关键。在本研究中,根据GenBank上公布的PCV3 DNA线性序列,使用SnapGene基因编辑软件对该序列进行重排,重排后在该线性DNA末端添加dIII限制性内切酶位点,使两端具有相同的限制性内切酶位点。在此基础上,通过基因合成、PCR扩增、DNA纯化等操作获得重排后的线性DNA,并进行酶切和连接以获得环化DNA。通过转染3D4/21细胞系获得PCV3感染性克隆。通过PCR、Western印迹和间接免疫荧光试验对获得的PCV3进行鉴定。结果表明,本研究成功获得了PCV3毒株。为进一步评估所获得的PCV3感染性克隆的致病性,本研究建立了感染PCV3的昆明小鼠动物模型。RT-PCR、Western印迹和免疫组化结果表明,PCV3可感染昆明小鼠的心肌和肺泡,但在其他组织中未检测到PCV3。上述研究表明,PCV3环状DNA可用于构建PCV3感染性克隆。本研究将为环状DNA病毒的构建提供一种新方法,并为PCV3疫苗的研究和发病机制奠定基础。