Infectious bovine viral diarrhea virus (BVDV) cDNA clones have been used for the expression of classical swine fever virus (CSFV) genes for immune prevention and control. However, can it be used for the expression of an allogenetic fragment? To answer this question, a BVDV chimeric virus expressing the spike (S) antigen fragment of porcine epidemic diarrhea virus (PEDV) was constructed. Antigen S_499-602 was inserted into pig-derived BVDV-2 infectious cDNA clone pASH28 in tandem by overlapping PCR, located between the seventh and eighth amino acids at the N-terminus of the capsid (C) protein of BVDV. Indirect immunofluorescence assay confirmed that the chimeric virus vASH-dS312 containing double S_499-602 sequences was successfully assembled, which could react with the monoclonal antibody (MAb) against BVDV E2 and PEDV S proteins. Further western blot analysis confirmed that the exogenous S_499-602 double protein could be stably expressed. Next, the chimeric virus vASH-dS312 was administered to BALB/C mice either orally or by intramuscular injection. The immunized mice were healthy and showed no signs of toxicity. IgG against BVDV and PEDV antibodies could be detected in the mice administered vASH-dS312 by intramuscular injection, which had neutralization activity against BVDV and PEDV. Thus, this study reported a new insertion site in the BVDV infectious cDNA clone that could successfully express an allogenetic antigen.

牛传染性腹泻病毒（BVDV）cDNA克隆已用于表达经典猪瘟病毒（CSFV）基因，用于免疫预防和控制。但是，它可以用于表达同基因片段吗？为了回答这个问题，构建了表达猪流行性腹泻病毒（PEDV）的刺突（S）抗原片段的BVDV嵌合病毒。将抗原S _499-602通过重叠PCR串联插入猪源性BVDV-2感染性cDNA克隆pASH28中，位于BVDV衣壳（C）蛋白N端的第七个和第八个氨基酸之间。间接免疫荧光分析证实嵌合病毒vASH-dS312含有双S _499-602序列已成功组装，可与针对BVDV E2和PEDV S蛋白的单克隆抗体（MAb）反应。进一步的蛋白质印迹分析证实，外源S _499-602双重蛋白可以稳定表达。接下来，将嵌合病毒vASH-dS312经口服或肌内注射给予BALB / C小鼠。免疫的小鼠是健康的，没有毒性迹象。在通过肌肉注射vASH-dS312的小鼠中可以检测到抗BVDV和PEDV抗体的IgG，该小鼠具有抗BVDV和PEDV的中和活性。因此，这项研究报告了BVDV传染性cDNA克隆中的一个新插入位点，该位点可以成功表达同种异体抗原。