人Boca病毒外壳蛋白VP2在杆状病毒中的表达及病毒样颗粒的构建  

The Expression of the Capsid Protein VP2 from Human Bocavirus Identified in Beijing and the Formation of Virus-Like Particles(VLPs) in Insect Cells

作　　者：赵林清[1] 钱渊[1] 丁雅馨[1] 朱汝南[1] 邓洁[1] 王芳[1] 孙宇[1] LiYan 

机构地区：[1]首都儿科研究所病毒研究室,北京100020 [2]加拿大人类和动物健康科学中心国家微生物研究室

出　　处：《病毒学报》2009年第5期333-338,共6页Chinese Journal of Virology

基　　金：国家自然科学基金(30872153);北京市科技新星(2006A63)

摘　　要：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。The aim of this study was to obtain the capsid protein VP2 of human bocavirus （HBoV） identified in Beijing recently and construct virus-like particles （VLPs） in insect cells for further study of this virus. The full-length VP2 gene of HBoV from BJ3722 was inserted into the baculovirus expression transfer vector （pFastBacTM1） to obtain the recombinant Bacmid, and generation of recombinant baculoviruses was followed by transfection of the recombinant Bacmid into insect cells. Then the recombinant VP2 protein was recognized by SDS-PAGE using Coomassie-blue staining and Western blot using hyper-immune serum against VP2 of HBoV from rabbit. The recombinant baculoviruses were harvested and amplified to gain large amounts of viruses with high titers to infect insect cells at a multiplicity of infection （MOI） of 0.5. After 7-10 days or 4-5 days of the infection, the supernatants of culture or the cell lysates treated with lysing solution were harvested, and ultracentrifuged twice through 40G sucrose cushion to obtain purified VLPs, which were followed by Western blot and IFA for VLPs＇ composition and specificity analysis, by electron microscopy for VLPs＇ morphologic structure. The recombinant VP2 protein with molecular weight of approximately 61 kD expressed in recombinant baculoviruses was recognized by SDS-PAGE using Coomassie-blue staining and Western blot. The presence of VP2 on VLPs was demonstrated by Western blot and IFA from samples collected during the purification of VLPs from the supernatants of culture or the cell lysates, and the expression of VP2 in insect ceils led to the formation of VLPs which formed the typical icosahedral appearance of parvoviruses with a diameter of approximately 20 nm. In conclusion, the recombinant baculoviruses were constructed, the HBoV VP2 protein was expressed in insect cells with high specific antigenicity and VLPs was formed successfully.

关 键 词：人Boca病毒 外壳蛋白VP2 重组杆状病毒 病毒样颗粒(VLPs) 

分 类 号：R373.1[医药卫生—病原生物学] Q78[医药卫生—基础医学]