Expression of Gn protein of severe fever with thrombocytopenia syndrome virus and its immunogenicity
Objective To express the Gn protein of severe fever with thrombocytopenia syndrome virus(SFTSV)through adeno-associated virus 9(AAV9)expression system and evaluate its immunogenicity.Methods SFTSV Gn gene was inserted into viral vector pAAV-CMV-FH and the recombinant plasmid was transfected into HEK293T cells to obtain recombinant virus AAV9-Gn.The expression of Gn protein was determined by immunofluorescence and Western blot.Eighteen female BALB/c mice were randomly divided into three groups:Mock group(serum-free DMEM),AAV9-GFP group(1 × 1011 vg)and AAV9-Gn group(1 × 1011 vg),all of which were injected intramuscularly into the right hind limb at a dose of 100 μL per mouse.The body mass,diet,behavior and mental state of mice in each group were monitored continuously for 21 d,and the change rate of body mass was calculated;At 2,4,8 and 16 weeks after immunization,the levels of SFTSV neutralizing antibody in serum of mice in each group were detected by fluorescent reduction neutralization test(FRNT),and the levels of specific IgG1 and IgG2a in serum of mice in AAV9-Gn group were detected by ELISA.Results After incubation with spe-cific antibody,Vero cells transfected with AAV9-Gn showed specific green fluorescence under fluorescence microscope,and had specific binding to mouse anti-SFTSV Gn monoclonal antibody,and the specific binding bands were found at a relative molecular mass of about 61 000.The body mass of the three groups showed an increasing trend,there was no significant difference between the three groups(F=0.158-2.621,P＞0.05),and the diet,behavior and mental state were nor-mal.At 2,4,8 and 16 weeks after immunization,the titer of SFTSV neutralizing antibody in serum of mice in AAV9-Gn group was significantly higher than that of Mock group and AAV9-GFP group(H=13.332-14.538,each P＜0.001),and the titer peak appeared at 8 weeks;The level of specific IgG1 in serum of mice was significantly higher than that of IgG2a(F=4.373-12.975,each P＜0.05)at different time points.Conclusion SFTSV Gn protein can be expressed correctly through AAV9 expression system,and has low toxicity to mice with good immunogenicity,which is expected to be a can-didate component of SFTSV vaccine.
Severe fever with thrombocytopenia syndrome virus(SFTSV)Gn proteinAdeno-associated virus 9(AAV9)Neutralizing antibodyIgG
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