Institute of Veterinary Immunology and Engineering, National Research Center of Engineering and Technology for Veterinary Biologicals, Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base of the Ministry of Science and Technology, Jiangsu Academy of Agricultural Sciences, 210014, Nanjing, Jiangsu, China.
Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, 225009, Yangzhou, Jiangsu, China.
BMC Vet Res. 2021 May 12;17(1):190. doi: 10.1186/s12917-021-02887-w.
Pseudorabies virus (PRV) is a preferred vector for recombinant vaccine construction. Previously, we generated a TK&gE-deleted PRV (PRV) based on a virulent PRV AH02LA strain. It was shown to be safe for 1-day-old piglets with maternal PRV antibodies and 4 ~ 5 week-old PRV antibody negative piglets and provide rapid and 100 % protection in weaned pigs against lethal challenge with the PRV variant strain. It suggests that PRV may be a promising live vaccine vector for construction of recombinant vaccine in pigs. However, insertion site, as a main factor, may affect foreign gene expression.
In this study, we constructed four recombinant PRV-S bacterial artificial chromosomes (BACs) carrying the same spike (S) expression cassette of a variant porcine epidemic diarrhea virus strain in different noncoding regions (UL11-10, UL35-36, UL46-27 or US2-1) from AH02LA BAC with TK, gE and gI deletion. The successful expression of S gene (UL11-10, UL35-36 and UL46-27) in recombinant viruses was confirmed by virus rescue, PCR, real-time PCR and indirect immunofluorescence. We observed higher S gene mRNA expression level in swine testicular cells infected with PRV-S(UL11-10)ΔTK/gE and PRV-S(UL35-36)ΔTK/gE compared to that of PRV-S(UL46-27)ΔTK/gE at 6 h post infection (P < 0.05). Moreover, at 12 h post infection, cells infected with PRV-S(UL11-10)ΔTK/gE exhibited higher S gene mRNA expression than those infected with PRV-S(UL35-36)ΔTK/gE (P = 0.097) and PRV-S(UL46-27)ΔTK/gE (P < 0.05). Recovered vectored mutant PRV-S (UL11-10, UL35-36 and UL46-27) exhibited similar growth kinetics to the parental virus (PRV).
This study focuses on identification of suitable sites for insertion of foreign genes in PRV genome, which laids a foundation for future development of recombinant PRV vaccines.
伪狂犬病病毒（PRV）是构建重组疫苗的首选载体。先前，我们基于一种强毒 PRV AH02LA 株构建了 TK&gE 缺失的 PRV（PRV）。研究表明，对于具有母源 PRV 抗体的 1 日龄仔猪和 PRV 抗体阴性的 4 至 5 周龄仔猪，该疫苗是安全的，在断奶猪中可快速、100%抵抗致死性 PRV 变异株攻毒。这表明 PRV 可能是构建猪重组疫苗的有前途的活疫苗载体。然而，插入位点作为一个主要因素，可能会影响外源基因的表达。
本研究在 AH02LA BAC 中构建了四个携带相同猪流行性腹泻病毒变异株刺突（S）表达盒的重组 PRV-S 细菌人工染色体（BAC），插入到 UL11-10、UL35-36、UL46-27 或 US2-1 等非编码区，这些 BAC 缺失了 TK、gE 和 gI。通过病毒拯救、PCR、实时 PCR 和间接免疫荧光证实了重组病毒中 S 基因（UL11-10、UL35-36 和 UL46-27）的成功表达。在感染猪睾丸细胞后 6 小时，与 PRV-S(UL46-27)ΔTK/gE 相比，感染 PRV-S(UL11-10)ΔTK/gE 和 PRV-S(UL35-36)ΔTK/gE 的细胞中 S 基因 mRNA 表达水平更高（P<0.05）。此外，在感染后 12 小时，感染 PRV-S(UL11-10)ΔTK/gE 的细胞中 S 基因 mRNA 表达水平高于感染 PRV-S(UL35-36)ΔTK/gE（P=0.097）和 PRV-S(UL46-27)ΔTK/gE（P<0.05）的细胞。回收的载体突变 PRV-S（UL11-10、UL35-36 和 UL46-27）与亲本病毒（PRV）具有相似的生长动力学。
本研究重点研究了在 PRV 基因组中插入外源基因的合适位置，为未来开发重组 PRV 疫苗奠定了基础。
