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构建多顺反子杆状病毒表面展示载体表达 PCV2 Cap(d41)蛋白及其在小鼠和猪中的免疫原性分析。

Construction of polycistronic baculovirus surface display vectors to express the PCV2 Cap(d41) protein and analysis of its immunogenicity in mice and swine.

机构信息

Department of Stomatology, Tung's Taichung MetroHarbor Hospital, Taichung, Taiwan.

Medical Biotechnology, National Chung Hsing University, Taichung, 402, Taiwan.

出版信息

Vet Res. 2020 Sep 9;51(1):112. doi: 10.1186/s13567-020-00836-3.

DOI:10.1186/s13567-020-00836-3
PMID:32907618
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7487469/
Abstract

To increase expression levels of the PCV2 Cap(d41) protein, novel baculovirus surface display vectors with multiple expression cassettes were constructed to create recombinant baculoviruses BacSC-Cap(d41), BacDD-2Cap(d41), BacDD-3Cap(d41), and BacDD-4Cap(d41). Our results reveal that the recombinant baculovirus BacDD-4Cap(d41) was able to express the highest levels of Cap(d41) protein. Optimum conditions for expressing the PCV2 Cap(d41) protein were determined, and our results show that 10 of Sf-9 infected with the recombinant baculovirus BacDD-4Cap(d41) at an MOI of 5 for 3 days showed the highest level of protein expression. Mice immunized with the 4Cap(d41) vaccine which was prepared from the recombinant baculovirus-infected cells (10) elicited higher ELISA titers compared to the Cap (d41) vaccine. The 4Cap(d41) vaccine could elicit anti-PCV2 neutralizing antibodies and IFN-γ in mice, as confirmed by virus neutralization test and IFN-γ ELISA. Moreover, the swine lymphocyte proliferative responses indicated that the 4Cap(d41) vaccine was able to induce a clear cellular immune response. Flow cytometry analysis showed that the percentage of CD4 T cells and CD4/CD8 ratio was increased significantly in SPF pigs immunized with the 4Cap(d41) vaccine. Importantly, the 4Cap(d41) vaccine induced an IFN-γ response, further confirming that its effect is through cellular immunity in SPF pigs. An in vivo challenge study revealed that the 4Cap(d41) and the commercial vaccine groups significantly reduce the viral load of vaccinated pigs as compared with the CE negative control group. Taken together, we have successfully developed a 4Cap(d41) vaccine that may be a potential subunit vaccine for preventing the disease associated with PCV2 infections.

摘要

为了提高 PCV2 Cap(d41) 蛋白的表达水平，构建了带有多个表达盒的新型杆状病毒表面展示载体，以创建重组杆状病毒 BacSC-Cap(d41)、BacDD-2Cap(d41)、BacDD-3Cap(d41)和 BacDD-4Cap(d41)。我们的结果表明，重组杆状病毒 BacDD-4Cap(d41)能够表达最高水平的 Cap(d41)蛋白。确定了表达 PCV2 Cap(d41)蛋白的最佳条件，结果表明，用重组杆状病毒 BacDD-4Cap(d41)以 MOI 为 5 感染 10 个 Sf-9 细胞，培养 3 天，蛋白表达水平最高。用重组杆状病毒感染细胞制备的 4Cap(d41)疫苗免疫小鼠，与 Cap(d41)疫苗相比，ELISA 效价更高。4Cap(d41)疫苗可在小鼠中诱导抗 PCV2 中和抗体和 IFN-γ，这通过病毒中和试验和 IFN-γ ELISA 得到证实。此外，猪淋巴细胞增殖反应表明，4Cap(d41)疫苗能够诱导明显的细胞免疫反应。流式细胞术分析表明，用 4Cap(d41)疫苗免疫 SPF 猪后，CD4 T 细胞的百分比和 CD4/CD8 比值显著增加。重要的是，4Cap(d41)疫苗诱导了 IFN-γ 反应，进一步证实其在 SPF 猪中的作用是通过细胞免疫。体内攻毒研究表明，与 CE 阴性对照组相比，4Cap(d41)疫苗和商品化疫苗组显著降低了接种猪的病毒载量。总之，我们成功开发了一种 4Cap(d41)疫苗，它可能是预防与 PCV2 感染相关疾病的潜在亚单位疫苗。

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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dd13/7487469/167d48743a1c/13567_2020_836_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/dd13/7487469/e1c12d4947c7/13567_2020_836_Fig7_HTML.jpg
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