表达猪细小病毒 VP2 蛋白和猪白细胞介素 6 的重组伪狂犬病病毒的特性。
Characterization of a recombinant pseudorabies virus expressing porcine parvovirus VP2 protein and porcine IL-6.
机构信息
College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengdong New District Longzi Lake#15, 450046, Zhengzhou, Henan Province, People's Republic of China.
Department of Life Science, Zhengzhou Normal University, Zhengzhou, 450044, Henan Province, People's Republic of China.
出版信息
Virol J. 2020 Feb 3;17(1):19. doi: 10.1186/s12985-020-1292-8.
BACKGROUND
Porcine parvovirus (PPV) and pseudorabies virus (PRV) are the important etiological agents of swine infectious diseases, resulting in huge economic losses to the Chinese swine industry. Interleukin-6 (IL-6) has the roles to support host immune response to infections as a pleiotropic cytokine. It is essential to construct a live attenuated vaccine-based recombinant PRV that expresses PPV VP2 protein and porcine IL-6 for prevention and control of PRV and PPV.
METHODS
The recombinant plasmid, pGVP2-IL6, was constructed by porcine IL-6 gene substituting for EGFP gene of the PRV transfer plasmid pGVP2-EGFP containing VP2 gene of PPV. Plasmid pGVP2-IL6 was transfected into swine testicle cells pre-infected with the virus rPRV-VP2-EGFP strain through homologous recombination and plaque purification to generate a recombinant virus rPRV-VP2-IL6. The recombinant PRV was further identified by PCR and DNA sequencing, and the expression of the VP2 protein and porcine IL-6 was analyzed by reverse transcription-PCR (RT-PCR) and Western blot. The virus titer was calculated according to Reed and Muench method. The immunogenicity of the recombinant virus was preliminarily evaluated in mice by intramuscular administration twice with the rPRV-VP2-IL6 at 4-week intervals.
RESULTS
A recombinant virus rPRV-VP2-IL6 was successfully constructed and confirmed in this study. The properties of rPRV-VP2-IL6 were similar to the parental virus HB98 in terms of growth curve, morphogenesis and virus plaque sizes, and rPRV-VP2-IL6 was proliferated in different cell types. It induced specific antibodies against PPV as well as a strong increase of PPV-specific lymphocyte proliferation responses in mice immunized with rPRV-VP2-IL6, and provided partial protection against the virulent PPV challenge. rPRV-VP2-IL6 also induced a high level of neutralizing antibodies against PRV, and significantly reduced the mortality rate of (1 of 10) following virulent PRV challenge compared with the control (10 of 10).
CONCLUSIONS
The recombinant rPRV-VP2-IL6 might be a potential candidate vaccine against PRV and PPV infections in pigs.
背景
猪细小病毒（PPV）和伪狂犬病病毒（PRV）是猪传染病的重要病原体，给中国的养猪业造成了巨大的经济损失。白细胞介素 6（IL-6）作为一种多效细胞因子，具有支持宿主对感染的免疫反应的作用。构建表达 PPV VP2 蛋白和猪 IL-6 的基于活减毒疫苗的重组 PRV 对于防控 PRV 和 PPV 至关重要。
方法
通过用 PPV 的 VP2 基因替换 PRV 转移质粒 pGVP2-EGFP 中的 EGFP 基因，构建了含有 PPV VP2 基因的猪 IL-6 基因的重组质粒 pGVP2-IL6。将质粒 pGVP2-IL6 通过同源重组和蚀斑纯化转染到预先感染病毒 rPRV-VP2-EGFP 株的猪睾丸细胞中，产生重组病毒 rPRV-VP2-IL6。通过 PCR 和 DNA 测序进一步鉴定重组 PRV，通过反转录-PCR（RT-PCR）和 Western blot 分析 VP2 蛋白和猪 IL-6 的表达。根据 Reed 和 Muench 方法计算病毒滴度。通过间隔 4 周两次肌肉注射 rPRV-VP2-IL6，初步评估重组病毒在小鼠中的免疫原性。
结果
本研究成功构建并证实了重组病毒 rPRV-VP2-IL6。rPRV-VP2-IL6 的特性与亲本病毒 HB98 在生长曲线、形态发生和病毒蚀斑大小方面相似，并且 rPRV-VP2-IL6 在不同的细胞类型中增殖。rPRV-VP2-IL6 免疫小鼠可诱导针对 PPV 的特异性抗体，并强烈增加 PPV 特异性淋巴细胞增殖反应，对强毒 PPV 攻毒提供部分保护。rPRV-VP2-IL6 还诱导了针对 PRV 的高水平中和抗体，并显著降低了强毒 PRV 攻毒后（10 只中的 1 只）的死亡率，与对照组（10 只中的 10 只）相比。
结论
重组 rPRV-VP2-IL6 可能是预防猪 PRV 和 PPV 感染的潜在候选疫苗。
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