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为了得到结构与功能更接近于天然病毒的人Boca病毒(HBoV)主要外壳蛋白VP2,将来源于北京急性呼吸道感染患儿标本(BJ3722)的HBoV VP2基因编码区克隆至杆状病毒表达转移载体(pFastBac1),通过转座反应获得重组Bacmid DNA,以这种DNA转染昆虫细胞Sf9。收获重组病毒及其昆虫细胞培养物,经SDS-PAGE并应用兔抗HBoV VP2高价免疫血清进行Western blot,以检测所表达的VP2蛋白。昆虫细胞经重组病毒感染后7~10d细胞完全裂解时,收获培养上清(VLPs-A),或在感染后4~5d细胞裂解前收获细胞并将其裂解(VLPs-B),经两次40%的蔗糖垫超速离心,所得的样品经Western blot和间接免疫荧光方法(IFA)检测,以确定VLPs的组分,然后应用免疫电镜观察病毒样颗粒的形态结构。通过考马斯亮蓝染色和Western blot检测,均可检测到分子量约61kD的目的蛋白;通过IFA在重组杆状病毒感染的Sf9细胞中检测到特异性荧光,表明HBoV VP2蛋白得到了表达并具有抗原特异性。纯化后的样品在电镜下可见类似于天然细小病毒B19的直径20nm左右的具有空壳结构的球形颗粒。本研究成功构建了HBoV VP2重组杆状病毒,得到了具有抗原特异性的HBoV VP2蛋白表达并形成了HBoV的VLPs。
In order to obtain VP2, the major coat protein of human Boca virus (HBoV), whose structure and function are more similar to that of natural virus, the coding region of HBoV VP2 gene from Beijing children with acute respiratory infection (BJ3722) was cloned into the baculovirus expression transfer vector pFastBac1). Recombinant Bacmid DNA was obtained by transposition reaction, and insect cells Sf9 were transfected with this DNA. Recombinant virus and insect cell cultures were harvested and subjected to Western blot by SDS-PAGE and Western blot with rabbit anti-HBoV VP2 high-titer immune serum to detect the expressed VP2 protein. When the cells were completely lysed after 7-10 days after infection with recombinant virus, the culture supernatants (VLPs-A) were harvested or cells were harvested and lysed (VLPs-B) 4 to 5 days after infection, The supernatant was centrifuged at 40% sucrose and the samples were detected by Western blot and indirect immunofluorescence (IFA) to determine the components of VLPs. The morphology of virus-like particles was observed by immunoelectron microscopy. The target protein with a molecular weight of about 61 kD was detected by Coomassie blue staining and Western blot. Specific fluorescence was detected by IFA in recombinant baculovirus-infected Sf9 cells, indicating that HBoV VP2 protein was expressed and had antigen specificity Sex. The purified sample shows spherical particles with a hollow shell structure with a diameter of about 20 nm similar to the native parvovirus B19 under the electron microscope. In this study, HBoV VP2 recombinant baculovirus was successfully constructed, and antigen-specific HBoV VP2 protein expression was obtained and HBoV VLPs were formed.