【作者】 王泽鋆；

【导师】 徐葛林； 李天成；

【作者基本信息】 武汉生物制品研究所 ， 免疫学， 2010， 硕士

【摘要】 2005年，瑞典学者首次报道在呼吸道疾病患者鼻咽分泌物中发现人博卡病毒1，随后，人博卡病毒2和人博卡病毒3又分别在急性迟缓型麻痹患儿和腹泻患儿的粪便中被相继发现。人博卡病毒是一类无包膜的单链线状DNA病毒，暂时划归为细小病毒科、细小病毒亚科、博卡病毒属。由于缺乏有效的体外细胞培养体系和动物模型，人博卡病毒与人类呼吸道或胃肠道疾病的临床关联尚且未知。因此，使用基因工程方法大量制备人博卡病毒抗原，建立相应的血清学检测方法，将有助于解开这些疑问。本研究使用昆虫细胞和杆状病毒表达载体系统表达人博卡病毒1、2和3衣壳蛋白VP2基因，重组衣壳蛋白VP2部可以自行组装成病毒样颗粒，使用纯化的人博卡病毒样颗粒建立酶联免疫吸附试验，检测200份西安地区健康婴儿、176份北京地区健康人血清中人博卡病毒1、2和3的IgG和IgM水平，以及373份日本福冈地区健康人血清中人博卡病毒1的IgG水平。电泳及透射电镜结果显示，大量分子量约为60．5kD的蛋白释放入培养上清中，重组蛋白可以自行组装成不含核酸的空心病毒样颗粒，这些病毒样颗粒平均直径为22nm，颗粒密度约为1．31g／ml。人博卡病毒血清流行病学结果显示，婴儿中人博卡病毒1、2和3的IgG阳性率分别为20．0％、14．0％和13．5％；而10岁以上人群中人博卡病毒1、2和3的IgG阳性率升高至75．6％、44．3％和42．0％；但是日本福冈地区人博卡病毒1的IgG阳性率超过80％。与IgG流行趋势相反，婴儿中人博卡病毒的IgM阳性率明显高于成年人群。昆虫细胞和杆状病毒表达载体系统可以有效表达人博卡病毒1、2和3衣壳蛋白VP2，并自行组装成病毒样颗粒。人博卡病毒在普通人群中广泛流行，成年人群中人博卡病毒的IgG的阳性率很高。更多还原

【Abstract】 Following the first indentification of HBoVl in nasopharyngeal secretion of a child with respiratory problems in 2005, HBoV2 and HBoV3 have recently been characterized in fecal samples from children with non-polio acute flaccid paralysis and diarrhea. Human bocaviruses (HBoVs) are small, non-enveloped, single-stranded DNA viruses, which have been provisionally classified in the family Parvoviridae, subfamily Parvovirinae, genus Bocavirus.Since no in vitro cell culture systems and animal models are available, whatever role HBoVs plays in respiratory infections and gastrointestinal illness remains uncertain. However, many of these problems will likely be solved within the nearestly years by production of recombinant antigens enabling serology.In this study, the major capsid protein HBoVs-VP2 genes, expressed in insect cells by baculovirus expression vector system, produced virus-like particles (VLPs), which were served to develop an ELISA. The VLPs-based ELISA was used to screen for HBoVs-IgG&IgM in 376 serum specimens from Xi’an and Beijing in China and 373 serum specimens from Fukuoka in Japan.SDS-PAGE and Electron microscopy results revealed a molecular weight of 60.5kD was generated and effectively released to culture supernatant, the 60.5kDa protein self-assembled into VLPs with a buoyant density of 1.31 g/cm3 and a diameter of 22nm. By the VLPs-based ELISA, 20%, 14% and 13.5% of serum specimens from infants, respectively, were found to be HBoVl, HBoV2 and HBoV3 IgG-positive; in contrast, the percentage of HBoVl, HBoV2 and HBoV3 IgG-positive increased to75.6%, 44.3% and 42% for adults; but the percentage of HBoVl IgG-positive is more than 80.0% in Fukuoka. Be different from IgG-seroprevalence, the percentage of HBoVs IgM-positive is higher in infants.The baculovirus expression vector system is well established for production of HBoVs capsid protein which could be self-assembled into VLPs. Preliminary seroepidemiologic data suggest that HBoVs are widely circulating in human populations and HBoVs infection is common during childhood.更多还原