表达猪圆环病毒2d型Cap蛋白的重组猪伪狂犬病病毒的构建

doi:10.16431/j.cnki.1671-7236.2025.05.031

Abstract

Abstract: 【Objective】 The purpose of this study was to obtain recombinant Porcine pseudorabies virus (PRV) expressing Porcine circovirus type 2d (PCV2d) Cap protein using homologous recombination technology and CRISPR/Cas9 gene editing technology,providing data support for the development of vaccines to prevent and control PCV2d and PRV infection.【Method】 According to the ORF2 gene sequence of the PCV2d KF1 strain,a pair of primers was designed and synthesized.The ORF2 gene was amplified by PCR from DNA extracted from the PCV2d KF1 strain,and was introduced into the PRV eukaryotic expression vector pG carrying the green fluorescent protein (EGFP) gene through BamH Ⅰ to construct a recombinant plasmid pG-PCV2d-EGFP.The recombinant virus was constructed by transfecting the plasmid pG-PCV2d-EGFP and DNA of the parental strain rPRV-gE^-/gI^-/TK^-PRV into ST cells using the transfection reagent ZLip2000,and the CRISPR/Cas9 EGFP knockout plasmid pX459-gRNA1-EZ-gRNA2 was used to knockout the EGFP gene of the recombinant virus.And the recombinant virus was identified by EGFP gene sequencing,PCR and Western blotting.【Result】 The recombinant virus strain rPRV-PCV2d-EGFP was obtained by eight rounds of the green fluorescent plaque screening.After EGFP gene was knocked out using the CRISPR/Cas9 EGFP knockout plasmid pX459-gRNA1-EZ-gRNA2,the recombinant virus rPRV-PCV2d was harvested after three rounds of the plaque screening without green fluorescent.The EGFP gene of recombinant virus rPRV-PCV2d was sequenced,and the results showed that EGFP gene was truncated by 1 251 bp.The results of PCR and Western blotting showed that the ORF2 gene of rPRV-PCV2d could be transcribed and expressed in ST cells.【Conclusion】 The recombinant virus rPRV-PCV2d expressing Cap protein of PCV2d was successfully constructed,which laid a foundation for further development of recombinant live vector vaccine.

Key words: Porcine circovirus type 2; Porcine pseudorabies virus; recombinant virus live vector vaccine

CLC Number:

S852.65⁺1

JIAO Xianqin, MA Xiao, TIAN Runbo, LIU Ying, MA Shijie, CHEN Hongying. Construction of Recombinant Porcine Pseudorabies Virus Expressing Cap Protein of Porcine Circovirus Type 2d[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(5): 2278-2286.

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Construction of Recombinant Porcine Pseudorabies Virus Expressing Cap Protein of Porcine Circovirus Type 2d

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