College of Veterinary Medicine, Jilin University, Changchun 130062, China; Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Institute of Military Veterinary, Academy of Military Medical Sciences, Changchun 130122, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China.
Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Institute of Military Veterinary, Academy of Military Medical Sciences, Changchun 130122, China.
Virus Res. 2015 Feb 2;197:59-66. doi: 10.1016/j.virusres.2014.12.015. Epub 2014 Dec 18.
Viral vectors are important vehicles in vaccine research. Avipoxviruses including fowlpox virus (FPV) play major roles in viral vaccine vector development for the prevention and therapy of human and other veterinary diseases due to their immunomodulatory effects and safety profile. Recently, we analyzed the genomic and proteomic backgrounds of the Chinese FPV282E4 strain. Based on analysis of the whole genome of FPV282E4, the FPV150 and FPV193 loci were chosen as insertion sites for foreign genes, and two shuttle vectors with a triple-gene expression cassette were designed and constructed. Homologous recombination between the FPV virus genome and sequences within the shuttle plasmids in infected cells was confirmed. The recombinants were obtained through several rounds of plaque purification using enhanced green fluorescent protein as a reporter and evaluated for the correct expression of foreign genes in vitro using RT-PCR, real-time PCR and Western blotting. Morphogenesis and growth kinetics were assayed via transmission electron microscopy and viral titering, respectively. Results showed that recombinant viruses were generated and correctly expressed foreign genes in CEF, BHK-21 and 293T cells. At least three different exogenous genes could be expressed simultaneously and stably over multiple passages. Additionally, the FPV150 mutation, FPV193 deletion and insertion of foreign genes did not affect the morphogenesis, replication and proliferation of recombinant viruses in cells. Our study contributes to the improvement of FPV vectors for multivalent vaccines.
病毒载体在疫苗研究中是重要的载体。禽痘病毒（FPV）包括鸡痘病毒（FPV），由于其免疫调节作用和安全性，在人类和其他兽医疾病的病毒疫苗载体开发中发挥主要作用。最近，我们分析了中国 FPV282E4 株的基因组和蛋白质组背景。基于对 FPV282E4 全基因组的分析，选择了 FPV150 和 FPV193 基因座作为外源基因的插入位点，并设计和构建了两个带有三基因表达盒的穿梭载体。在感染细胞中，FPV 病毒基因组与穿梭质粒内的序列之间发生了同源重组。通过使用增强型绿色荧光蛋白作为报告基因进行几轮噬斑纯化，获得了重组体，并通过 RT-PCR、实时 PCR 和 Western blot 体外评估了外源基因的正确表达。通过透射电子显微镜和病毒滴定分别测定形态发生和生长动力学。结果表明，重组病毒在 CEF、BHK-21 和 293T 细胞中生成并正确表达了外源基因。至少可以同时稳定表达三种不同的外源基因，并且可以在多个传代中稳定表达。此外，FPV150 突变、FPV193 删除和外源基因的插入不影响重组病毒在细胞中的形态发生、复制和增殖。我们的研究为多价疫苗的 FPV 载体的改进做出了贡献。
