表达非洲猪瘟病毒p54蛋白重组伪狂犬病病毒的构建及鉴定

doi:10.16431/j.cnki.1671-7236.2021.11.028

Abstract

Abstract: The objective of this study was to construct a recombinant Pseudorabies virus (PRV) with high expression of the p54 protein of African swine fever virus (ASFV). A universal transfer vector pCAGIG-TK_(l+r) containing homologous arm of PRV TK gene and green fluorescent protein (GFP) reporter gene was constructed by seamless cloning. The E183L gene was optimized and synthesized according to the gene sequence of China/2018/AnhuiXCGQ strain, and inserted into the universal transfer vector to construct the recombinant intermediate transfer plasmid pCAGIG-TK_(l+r)-p54. The recombinant plasmid was linearized by ScaⅠenzyme and then transfected into BHK-21 cells by lipofectamine. 6 h later, 0.1 MOI of PRV was infected. After the lesion appeared, the recombinant PRV was purified by plaque selection and PCR. Furthermore, the expression of foreign protein was detected by Western blotting and indirect immunofluorescence assay (IFA), and the genetic stability and proliferation characteristics of the recombinant virus were studied. The results showed that green fluorescence could be observed in BHK-21 cells transfected with pCAGIG-TK_(l+r)-p54 after 24 h, indicating that the recombinant plasmid was successfully transferred into BHK-21 cells. The purified recombinant virus expressed green fluorescent protein and contained ASFV E183L gene. Western blotting and IFA results showed that the recombinant PRV could express the p54 protein in BHK-21 cells, which showed a specific band at 26 ku, and had a specific reaction with ASFV positive serum, showing good immunogenicity. The ASFV E183L gene could be detected in BHK-21 cells after 20 successive passages, showing good genetic stability. One-step growth curve showed that there was no significant difference in the proliferation characteristics between the recombinant virus and the parental virus, and their highest viral titers were 10^7.34/0.1 mL and 10^7.61/0.1 mL, respectively. The insertion of foreign fragments did not affect the proliferation ability of the recombinant virus in cells. In this study, recombinant virus rPRV-p54 was successfully obtained, which could express ASFV p54 protein efficiently, and laid a foundation for the further study of the immunogenicity of p54 protein and the development of multigene recombinant PRV vector vaccine for ASFV.

Key words: African swine fever virus (ASFV); Pseudorabies virus (PRV); p54 protein

CLC Number:

S852.65⁺1

HE Xinglin, ZOU Zhong, GONG Wenxiao, ZHANG Yufei, LI Chengfei, XU Ting, CHEN Huanchun, JIN Meilin. Construction and Identification of Recombinant Pseudorabies Virus Expressing the p54 Protein of African Swine Fever Virus[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(11): 4182-4191.

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Construction and Identification of Recombinant Pseudorabies Virus Expressing the p54 Protein of African Swine Fever Virus

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