Preparation of porcine circovirus type 2 virus-like particles and its immunogenicity research.
Preparation of porcine circovirus type 2 virus-like particles and its immunogenicity research.
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DOI：
10.16656/j.issn.1673-4696.2021.0138
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发表时间：
2021-01-01
期刊：
影响因子：
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通讯作者：
Liu, Chang-Ming
中科院分区：
文献类型：
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作者：
Bian, Hai-Qiao;Huang, Li-Ping;Liu, Chang-Ming
利用真核细胞表达系统表达猪圆环病毒2型（PCV 2）Cap蛋白，研究病毒样颗粒（VLP）的形成及其免疫原性。以p CAGGS为表达载体构建重组质粒p CAGGS-Cap-Flag，瞬时转染293 T细胞，表达的Cap蛋白通过Flag标签纯化。电镜观察重组Cap蛋白形成的VLP形态，纯化的重组Cap蛋白可形成直径约17 nm的VLP，与天然PCV 2病毒颗粒一致。经SDS-PAGE分析，重组Cap蛋白分子量为28.7ku，与预期分子量相符。Western-blot结果表明，重组Cap蛋白与PCV 2特异性单克隆抗体具有良好的免疫应答，捕获ELISA结果表明，重组Cap蛋白组装的VLP具有与纯化的天然PCV 2相似的抗原活性。分别用制备的VLP和经典的PCV 2灭活疫苗免疫小鼠。两种疫苗均能诱导产生良好的PCV 2抗体应答，免疫小鼠的抗体效价达到12 800，是灭活疫苗的2倍。采用PCR方法检测免疫后和攻毒后小鼠肺组织中PCV 2核酸。结果显示，VLP免疫组和PCV 2灭活疫苗免疫组均未检出PCV 2核酸，判定为100%保护。本研究利用真核细胞表达系统制备的PCV 2-VLPs具有与天然病毒颗粒相似的形态，并在小鼠体内诱导了良好的免疫应答。它可以用于开发新的PCV 2疫苗。
Porcine circovirus type 2(PCV2) recombinant Cap protein was expressed using an eukaryotic cell expression system to explore the formation and immunogenicity of virus-like particles(VLPs). The recombinant plasmid p CAGGS-Cap-Flag was constructed with p CAGGS as the expression vector and transfected transiently into 293 T cells, and the expressed Cap protein was then purified by the Flag tag. Observation of the VLPs morphology formed by the recombinant Cap protein by electron microscopy showed that the purified recombinant Cap protein could form VLPs with a diameter of approximately 17 nm, which was consistent with natural PCV2 virus particles. The recombinant Cap protein was 28.7 ku and the molecular weight was in line with expectations by SDS-PAGE. A good immune response of the recombinant Cap protein with PCV2 specific monoclonal antibodies was shown by Western-blot.It was found that the VLPs assembled by the recombinant Cap protein had the similar antigen activity as the purified natural PCV2 by capture ELISA. The prepared VLPs and the classic PCV2 inactivated vaccine were used to immunize mice respectively. Both of the two vaccines could induce a good PCV2 antibody response, and the antibody titers of mice immunized with VLPs was tested by IPMA to reach up to 12 800, which was twice the antibody titer of mice immunized with PCV2 inactivated vaccine. The PCV2 nucleic acid in the lung tissue of the mice was tested by PCR after immunization and challenge. The results showed that neither the VLPs immunized group nor the PCV2 inactivated vaccine immunized group had PCV2 nucleic acid detected, and it was judged to be 100% protected. PCV2-VLPs prepared by the eukaryotic cell expression system in this study had a similar morphology to that of natural virus particles and induced a good immune response in mice. It may be used for the development of new PCV2 vaccines.