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只能在骨髓和胎儿肝脏的红细胞前体中复制。这种对宿主细胞分化的极度依赖导致病毒材料有限，使骨髓移植接受者、溶血性贫血患者和孕妇等高危人群患者难以早期诊断感染和接种疫苗。在本研究中，我们利用单克隆抗体和位点特异性合成多肽，确定了参与病毒中和的病毒衣壳蛋白位点，并成功观察到VP-2在哺乳动物细胞和大肠杆菌中的表达，该抗原可作为以下血清学诊断和疫苗接种的抗原1。在体外培养的人骨髓细胞受病毒攻击后，获得了一种保护CFU-e减少的单克隆抗体BE11。利用VP-2的氨基酸序列推断出的10个合成多肽确定了该单克隆抗体的抗原表位。BE11只反应了VP-2.2 n端328 ~ 344个氨基酸的肽段。转染含有SV40晚期启动子和VP-2编码区的质粒后，COS猴细胞核内病毒蛋白积累量达到58 kd。这说明VP-2.3中存在核定位信号。VP-2在大肠杆菌中的基因表达。将VP-2的编码区克隆到大肠杆菌外膜蛋白信号肽编码区下游，构建质粒。在大肠杆菌质周空间成功表达了一个58kd具有VP-2抗原性的蛋白。我们现在正在使用这种重组蛋白建立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.