Analysis of mode of gene expression of human parvovirus and application of the recombinant viral protein in clinical diagnosis

人细小病毒B19在体外只能在骨髓和胎肝中的红系前体细胞中复制。这种对宿主细胞分化的极端依赖性导致病毒的材料有限，并且使得感染的早期诊断和高危群体患者如骨髓移植受者、患有溶血性贫血的患者和孕妇的疫苗接种变得困难。在这项研究中，我们利用单克隆抗体和位点特异性合成多肽鉴定了参与病毒中和的病毒衣壳蛋白位点，并成功地在哺乳动物细胞和大肠杆菌中表达了VP-2，为VP-2的血清学诊断和疫苗接种提供了抗原。2.获得了抗病毒单克隆抗体BE 11，该单克隆抗体对体外培养的人骨髓细胞经病毒攻击后CFU-e的下降具有保护作用。利用VP-2氨基酸序列推导的10个合成多肽确定了该单克隆抗体的表位位点。2. VP-2在哺乳动物细胞中的基因表达用含有VP-2编码区和SV 40晚期启动子的质粒转染COS猴细胞，在细胞核中观察到58 kd的病毒蛋白积累。这表明VP-2中存在核定位信号。3. VP-2基因在大肠杆菌中的表达。将VP-2的编码区克隆到大肠杆菌外膜蛋白信号肽编码区的下游，构建质粒。在大肠杆菌的周质空间中成功表达了具有VP-2抗原性的58 kd蛋白。我们现在正在建立使用该重组蛋白进行临床诊断的检测系统。

Human parvovirus B19 can replicate only in erythroid precursors in bone marrow and fetal liver in vitro. This extremely dependence on host cell differentiation causes the limited materials of the virus and makes difficult the early diagnosis of the infection and vaccination for high risk group patient such as recipient of bone marrow transplantation, patients suffering from hemolytic anemias and pregnant women. In this study, we identified the site of viral capsid protein which involved the neutralization of the virus, using monoclonal antibody and site specific synthetic polypeptides, and also successfully observed the expression of VP-2 in mammalian cells and E.coli which could be used for the antigen for the serological diagnosis and vaccination as follow.1.Identification of neutralization responsive site in VP-2 We obtained a monoclonal antibody BE11 which protected decrease of CFU-e in cultured human bone marrow cells challenged by the virus in vitro. The site of epitope of this monoclonal antibody was determined using 10 synthetic polypeptides deduced by the amino acid sequence of VP-2. BE11 only reacted a peptide which represented from 328th to 344th amino acid from the N-terminal of VP-2.2.Gene expression of VP-2 in mammalian cells Accumulation of 58 kd of viral protein in the nucleus was observed in COS monkey cells transfected a plasmid containing SV40 late promoter and coding region of VP-2. This suggests the existence of nuclear localization signal in VP-2.3.Gene expression of VP-2 in E.coli. A plasmid was constructed by cloning the coding region of VP-2 into the downstream of the coding region of signal peptide of outer membrane protein of E.coli. A 58 kd of protein having the antigenicity of VP-2 was successfully expressed in the periplasmic space of E.coli. We are now setting up the assay system for clinical diagnosis using this recombinant protein.