病毒学报

为了解纯化后灭活发热伴血小板减少综合征病毒(Severe fever with thrombocytopenia syndrome bunyavirus,SFTSV)的免疫原性,本研究通过超滤浓缩和分子筛层析相结合的方法对灭活的SFTSV进行纯化,采用Western blot和SDS-PAGE对灭活病毒的纯化样品进行分析和鉴定,采用双抗夹心ELISA方法对其中糖蛋白(Glycoprotein,GP)和核蛋白(Nucleoprotein,NP)的抗原含量进行检测。浓缩纯化的SFTSV灭活病毒,经电镜观察并通过BCA法测定其总蛋白浓度。然后,将纯化的灭活SFTSV按两种不同的免疫程序免疫新西兰兔,评价免疫原性和比较免疫效果。结果显示,SFTSV灭活并超滤浓缩后,分子筛层析可观察到两个洗脱峰,其中之一为灭活病毒颗粒,SDS-PAGE、Western blot和ELISA法分析均可检测到GP和NP抗原,而另一洗脱峰主要成分则为NP。浓缩后的纯化病毒纯度达90%以上,总蛋白浓度约为1.1mg/mL,且具有典型的布尼亚病毒电镜形态。纯化的SFTSV灭活病毒免疫实验动物后,可在血清中检出高滴度病毒特异性IgG和中和抗体,但抗体滴度受免疫程序的影响,0d、14d和28d天三针程序免疫动物的效果明显优于0d、7d和28d免疫程序组。本研究显示了灭活SFTSV培养液中除完整病毒颗粒外,还含有大量游离的NP,经分子筛层析纯化可获得高纯度灭活病毒,同时明确了纯化的SFTSV灭活病毒具有良好的免疫原性,可诱导产生高滴度中和抗体和病毒特异IgG抗体,可为灭活病毒疫苗的进一步研究提供重要的线索。

To understand the immunogenicity of purified inactivated severe fever with thrombocytopenia syndrome bunyavirus(SFTSV),concentration by ultrafiltration as well as molecular-sieve chromatography(MSC)were used for purification of inactivated SFTSVs.Inactivated viruses in purified samples were analyzed and identified by western blotting and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),the glycoprotein(GP)and nucleoprotein(NP)antigen titers of which were detected using a double-sandwich enzyme-linked immunosorbent assay(ELISA).Purified inactivated SFTSVs were enriched and observed by electron microscopy,and the total protein concentration detected using the bicinchoninic acid assay.Purified inactivated SFTSVs were applied to New Zealand rabbits via two immunization programs to evaluate immunogenicity and to compare the immune effect.After SFTSVs were inactivated and concentrated by ultrafiltration,MSC revealed two typical elution peaks.The sample of one peak was identified as inactivated virions,in which GP and NP were detected by SDS-PAGE,western blotting and ELISA.Main component of the other peak was NP.After concentration by ultrafiltration,purified inactivated SFTSVs with purity>90%and total protein concentration of 1.1mg/mL were obtained,and the typical electron microscopy of bunyavirus was observed.In the sera of animals immunized with purified inactivated SFTSVs,SFTSV-specific IgG antibody and neutralizing antibody were detected at high titers.However,antibody titers were affected by the immunization program.Effect of immunization on days 0,14 and 28was significantly better than that on days 0,7and 28.Our work revealed that cultivation of SFTSVs contained intact virus particles and large amounts of free NP.Using MSC,purified inactivated SFTSVs of high purity could be obtained.Purified inactivated SFTSVs induced high titers of neutralizing antibody and virus-specific IgG antibody showing satisfactory immunogenicity,which provides important clues for further study on a vaccine for the inactivated virus.