发热伴血小板减少综合征病毒Gn蛋白的表达及其免疫原性

Expression of Gn protein of severe fever with thrombocytopenia syndrome virus and its immunogenicity

目的通过9型腺相关病毒(adeno-associated virus 9,AAV9)系统表达发热伴血小板减少综合征病毒(severe fever with thrombocytopenia syndrome virus,SFTSV)Gn蛋白,并评价其免疫原性。方法将SFTSV Gn基因重组至病毒载体pAAV-CMV-FH,将构建的重组质粒转染HEK293T细胞,获得重组病毒AAV9-Gn;免疫荧光和Western blot法鉴定Gn蛋白的表达情况。将18只雌性BALB/c小鼠随机分为Mock组(无血清DMEM)、AAV9-GFP组(1×10^(11)vg)和AAV9-Gn组(1×10^(11)vg),各组均经小鼠右后肢肌内注射,100μL/只。连续21 d监测各组小鼠体质量、饮食、行为及精神状态,并计算体质量变化率;于免疫后2、4、8、16周,采用荧光灶减少中和试验(fluorescent reduction neutralization test,FRNT)检测各组小鼠血清中SFTSV中和抗体水平,ELISA法检测AAV9-Gn组小鼠血清中特异性IgG1和IgG2a抗体水平。结果与特异性抗体孵育显色后,转染AAV9-Gn的Vero细胞于荧光显微镜下可见特异性绿色荧光,且可与小鼠抗SFTSV Gn单克隆抗体发生特异性结合,并于相对分子质量约61000处可见特异性结合条带。3组小鼠体质量均呈增长趋势,组间差异无统计学意义(F=0.158~2.621,P>0.05),且饮食、行为及精神状态均表现正常。免疫后2、4、8和16周,AAV9-Gn组小鼠血清中的SFTSV中和抗体效价均明显高于Mock组和AAV9-GFP组(H=13.332~14.538,P均<0.001),且效价峰值出现在第8周;不同时间点小鼠血清中特异性IgG1抗体水平均明显高于IgG2a(F=4.373~12.975,P均<0.05)。结论通过AAV9表达系统可正确表达SFTSV Gn蛋白,且对小鼠毒性较低,并具有良好的免疫原性,有望作为SFTSV疫苗的候选成分。

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 fernale BALB/c mice were randomly divided into three groups:Mock group(serum-free DMEM),AAV9-GFP group(1×10^(11)vg)and AAV9-Gn group(1×10^(11)vg),all of which were injected intramuscularly into the right hind limb at a dose of 100μL 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 IgGl and IgG2a in serum of mice in AAV9-Gn group were detected by ELISA.Results After incubation with specific 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 61000.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 normal.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 candidate component of SFTSV vaccine.