杆状病毒表达系统表达BCoV纤突蛋白及其对小鼠的免疫原性

doi:10.11843/j.issn.0366-6964.2024.02.021

Abstract

Abstract: This study aimed to express the S protein of Bovine coronavirus (BCoV) and evaluate its immunogenicity in mice. The S protein of BCoV/SWUN/HXD-4/2021 strain was seclected as a template for codon optimization in insect cells. The complete S gene was synthesized and the recombinant plasmid pFastBac-Dual-S and Bacmid-S were constructed. The recombinant baculovirus rpFastBac-S was harvested by transfection of insect cells and identified by PCR, Western blot and indirect immunofluorescence assay. Baculovirus titers were determined. The effects of different infection doses (MOI=0.005, 0.05, 0.5, 1, 2) on protein expression were optimized, and the best infection condition was selected to express protein. The protein was purified by ultracentrifugation and immunized mice, and the immune effect was evaluated by indirect ELISA and neutralization test. After optimization, the S gene CAI (codon adaptation index) was adjusted from 0.39 to 0.87, and the average GC content was adjusted from 35.8% to 50.6%. Double enzyme digestion and PCR confirmed that the recombinant plasmid and the recombinant bacmid were successfully constructed. Sf9 cells were transfected with 5 μg Bacmid-S and the cytopathic effect tended to be steady after the fourth cell passage. Typical cytopathic effect appeared 20 h after baculovirus infection. The recombinant bacmid-S was harvested and identified. The results of PCR electrophoresis showed that the recombinant bacmid carried the S target protein gene (size 4 108 bp). IFA showed that specific green fluorescence was observed in the experimental group. Western blot results showed that the recombinant baculovirus rpFastBac-S protein was the S target protein, and the target size was about 250 ku. When the MOI was 0.5, the protein expression level was the highest. The mice were immunized with 50 μg protein, 20 μg CpG adjuvant and equal volume of MF59 adjuvant by intramuscular injection. Indirect ELISA results showed that the mice immunized with the protein produced IgG specific antibody, and the antibody titer was higher than that of the control group (P<0.001), and the highest antibody titer was 1∶12 800. The results of neutralization test showed that the highest neutralization titer of mouse serum was 1∶224, and the average neutralization titer of experimental group was higher than that of inactivated BCoV group, but the difference was not statistically significant (P>0.05). In conclusion, the S protein of BCoV was expressed by baculovirus expression system, and the immune protective effect of S protein was evaluated, which provided a theoretical foundation for the subsequent research of bovine coronavirus vaccine.

Key words: bovine coronavirus, S protein, baculovirus expression system, immunogenicity

CLC Number:

S852.659.6

YU Qisheng, ZHU Qing, ZHOU Qun, SONG Xin, ZHANG Jiaqi, CHEN Taoyun, XU Lin, ZHANG Chaohui, ZHANG Bin. Expression of BCoV Spike Protein by Baculovirus Expression System and Its Immunogenicity in Mice[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 640-648.

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Expression of BCoV Spike Protein by Baculovirus Expression System and Its Immunogenicity in Mice

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