中国人丙型肝炎病毒基因组3′端非编码区的研究

目的分析中国丙型肝炎病人ＨＣＶ基因组３′端非编码区（３′ＮＣＲ），以促进对ＨＣＶ基因组复制机制的研究。方法采用两种方法，从上海地区感染ＨＣＶ的病人血清中，扩增获得ＨＣＶ基因组３′端非编码区：一是用套式ＰＣＲ直接扩增，二是先分别获得ＨＣＶ３′ＮＣＲ的前半部分和后半部分，再将两片段进行融合ＰＣＲ。ＰＣＲ产物进行测序后作同源性分析。在此基础上，建立了针对３′端非编码区的ＲＴＰＣＲ方法，并与基于５′端非编码区的ＲＴＰＣＲ方法检测ＨＣＶＲＮＡ的特异性和灵敏度比较。结果序列分析表明，中国丙型肝炎病人ＨＣＶ基因组３′非编码区由４部分组成：高度变异区、Ｐｏｌｙ（Ｕ）区、Ｐｏｌｙ（Ｕ／Ｃ）区和９８碱基区。同源性分析显示，９８碱基区在不同分离株间高度保守并与国外报道株一致，而Ｐｏｌｙ（ＵＵＣ）区存在较大差异。３′端非编码区和５′非编码区ＲＴＰＣＲ检测血清ＨＣＶＲＮＡ有较高符合率（９５％）。结论ＨＣＶ基因组３′端非编码区的３′末端（９８碱基），在不同分离株间的高度保守性提示，该区在ＨＣＶ基因复制中起重要作用。基于３′非编码区的ＲＴＰＣＲ方法，将有助于ＨＣＶ感染的诊断。

Study of the 3noncoding region of Chinese hepatitis C virus genome

Objective To analyze the 3 noncoding region (3NCR) of HCV genome from Chinese hepatitis C patients so as to facilitate further study of mechanism of HCV gene replication. Methods Two different strategies were employed to amplify the fulllength of the 3 noncoding region of HCV genome from sera of HCV infected patients in Shanghai area: one was to amplify the fulllength fragment directly by nested PCR and the other amplify two overlapping fragments. The PCR products were further analyzed by sequencing and nucleotide alignments. A HCV genome 3NCR based RTPCR was developed and its specificity and sensitivity for HCV RNA detection in sera was compared with the established 5NCR based RTPCR. Results Sequence analysis showed that Chinese HCV genomic 3 NCR consists of three parts: the 5 region, poly (UUC) tract and the 98base region. Sequence alignments revealed that, while the 98base regions were completely conserved in different isolates and were identical to the reported sequences, the poly (UUC) region shared highly diversities. A high degree of concordance(95%) between the 3NCR and 5NCR RTPCR for detection of HCV RNA in sera was found. Conclusion The high conservation at the 3 NCR(98 bases) of HCV genome among different isolates indicated that this region may be critical for HCV gene replication The 3NCR based RTPCR may be a useful addition to available systems to diagnosis HCV infection.