基于E2蛋白BVDV-1病毒样颗粒的构建及对小鼠的免疫效力评估

doi:10.11843/j.issn.0366-6964.2023.12.024

Abstract

Abstract: This study aimed to construct the BVDV-1(bovine viral diarrhea virus 1) E2 virus-like particles (VLP) by the insect cell-baculovirus expression system and to evaluate its immune efficacy in mice. The E2 gene sequence of BVDV-1 SWU-Z6 strain was optimized and synthesized according to the codon bias of insect cells, and used to generate the construct of the BVDV-1 E2 VLPs for insect cell-baculovirus expression system. The expression of E2 protein in recombinant baculovirus was validated by the indirect immunofluorescence (IFA) and Western blot, and the assembly of VLPs was evaluated through electron microscope. The VLPs were then purified using sucrose density centrifugation. Mice were immunized with different doses of VLPs plus MF59 adjuvant and CpG-ODN immune enhancer by intramuscular injection, with BVDV commercial inactivated vaccine group and PBS blank as control group. Four weeks after booster immunization, the mice were challenged using BVDV-1 SWU-Z6 strain via gavage. The immune protection effect of BVDV-1 E2 VLPs was evaluated based on weight changes and viral load in feces of mice. The recombinant plasmid pFast Dual BVDV-1 E2 was constructed by inserting the optimized E2 gene and verified by restriction endonuclease analysis and sequencing. After transformed into DH10.Bac competent cells, the recombinant baculovirus Baculo-BVDV-1 E2 were extracted and transfected into SF9 insect cells. The BVDV-1 E2 VLPs with diameter about 80-100 nm were observed by electron microscope. The E2 protein expressed by the recombinant baculovirus was further confirmed by IFA and Western blot analysis, exhibiting a good biological activity in vitro. The antibody titer in group injected with 50 μg of VLPs was about 1∶10 000 2 weeks after first immunization, and reached 1∶100 000 2 weeks after enhanced immunization by ELSIA. After the challenge with SWU-Z6 strain, the immunized mice showed weight loss 7 days after infection before beginning to recover. No virus was detected in feces collected on day 10 after infection. While the mice group injected 50 μg of BVDV-1 E2 VLPs twice showed no weight loss and virus replication in the digestive tract in comparison with the non-immunized mice. The BVDV-1 E2 VLPs expressed by the insect cell-baculovirus expression system induce an efficient BVDV-specific humoral immune responses in mice, and prevent the BVDV-1 SWU-Z6 strain infection.

Key words: bovine viral diarrhea virus, E2 protein, baculovirus, virus-like particles, immunogenicity

CLC Number:

S852.659.6

ZHANG Jiaqi, JIA Xining, ZHOU Qun, SONG Xin, ZHU Chenxi, LI Gege, ZHANG Bin. Construction of BVDV-1 Virus-like Particles based on E2 Protein and Evaluation of Its Immune Efficacy in Mice[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5143-5153.

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Construction of BVDV-1 Virus-like Particles based on E2 Protein and Evaluation of Its Immune Efficacy in Mice

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