Hsu Chao-Yu, Jang Yun, Huang Wei-Ru, Wang Chi-Young, Wen Hsiao-Wei, Tsai Pei-Chien, Yang Cheng-Yao, Munir Muhammad, Liu Hung-Jen
Department of Medical Research, Tungs' Taichung Metroharbor Hospital, Taichung 435, Taiwan.
Ph.D. Program in Translational Medicine, National Chung Hsing University, Taichung 402, Taiwan.
Vaccines (Basel). 2023 Oct 31;11(11):1666. doi: 10.3390/vaccines11111666.
To simultaneously express and improve expression levels of multiple viral proteins of a porcine reproductive and respiratory syndrome virus (PRRSV), polycistronic baculovirus surface display vectors were constructed and characterized. We engineered polycistronic baculovirus surface display vectors, namely, pBacDual Display EGFP(BacDD)-2GP2-2GP4 and pBacDD-4GP5N34A/N51A (mtGP5), which simultaneously express and display the ectodomain of His-tagged GP2-gp64TM-CTD, His-tagged GP4-gp64TM-CTD, and His-tagged mtGP5-gp64TM-CTD fusion proteins of PRRSV on cell membrane of Sf-9 cells. Specific pathogen-free (SPF) pigs were administered intramuscularly in 2 doses at 21 and 35 days of age with genetic recombinant baculoviruses-infected cells. Our results revealed a high level of ELISA-specific antibodies, neutralizing antibodies, IL-4, and IFN-γ in SPF pigs immunized with the developed PRRSV subunit vaccine. To further assess the co-expression efficiency of different gene combinations, pBacDD-GP2-GP3-2GP4 and pBacDD-2mtGP5-2M constructs were designed for the co-expression of the ectodomain of His-tagged GP2-gp64TM-CTD, His-tagged GP3-gp64TM-CTD, and His-tagged GP4-gp64TM-CTD proteins as well as the ectodomain of His-tagged mtGP5-gp64TM-CTD and His-tagged M-gp64TM-CTD fusion proteins of PRRSV. To develop an ELISA assay for detecting antibodies against PRRSV proteins, the sequences encoding the ectodomain of the GP2, GP3, GP4, mtGP5, and M of PRRSV were amplified and subcloned into the pET32a vector and expressed in In this work, the optimum conditions for expressing PRRSV proteins were evaluated, and the results suggested that 4 × 10 of Sf-9 cells supplemented with 7% fetal bovine serum and infected with the recombinant baculoviruses at an MOI of 20 for three days showed a higher expression levels of the protein. Taken together, the polycistronic baculovirus surface display system is a useful tool to increase expression levels of viral proteins and to simultaneously express multiple viral proteins of PRRSV for the preparation of subunit vaccines.
为了同时表达和提高猪繁殖与呼吸综合征病毒（PRRSV）多种病毒蛋白的表达水平，构建并鉴定了多顺反子杆状病毒表面展示载体。我们构建了多顺反子杆状病毒表面展示载体，即pBacDual Display EGFP(BacDD)-2GP2-2GP4和pBacDD-4GP5N34A/N51A（mtGP5），它们能在Sf-9细胞细胞膜上同时表达和展示PRRSV的His标签化GP2-gp64跨膜区-羧基末端结构域、His标签化GP4-gp64跨膜区-羧基末端结构域以及His标签化mtGP5-gp64跨膜区-羧基末端结构域融合蛋白。在21日龄和35日龄时，给无特定病原体（SPF）猪分两剂肌肉注射基因重组杆状病毒感染的细胞。我们的结果显示，用所开发的PRRSV亚单位疫苗免疫的SPF猪体内产生了高水平的ELISA特异性抗体、中和抗体、白细胞介素-4和干扰素-γ。为了进一步评估不同基因组合的共表达效率，设计了pBacDD-GP2-GP3-2GP4和pBacDD-2mtGP5-2M构建体，用于共表达PRRSV的His标签化GP2-gp64跨膜区-羧基末端结构域、His标签化GP3-gp6至4跨膜区-羧基末端结构域以及His标签化GP4-gp64跨膜区-羧基末端结构域蛋白，以及PRRSV的His标签化mtGP5-gp64跨膜区-羧基末端结构域和His标签化M-gp64跨膜区-羧基末端结构域融合蛋白。为了开发一种检测抗PRRSV蛋白抗体的ELISA检测方法，扩增了编码PRRSV的GP2、GP3、GP4、mtGP5和M的胞外域的序列，并亚克隆到pET32a载体中并在其中表达。在这项工作中，评估了表达PRRSV蛋白的最佳条件，结果表明，4×10个补充有7%胎牛血清且以20的感染复数感染重组杆状病毒三天的Sf-9细胞显示出较高的蛋白表达水平。综上所述，多顺反子杆状病毒表面展示系统是提高病毒蛋白表达水平以及同时表达PRRSV多种病毒蛋白以制备亚单位疫苗的有用工具。
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