医学检验信息网 检验医学 2007-2-26 16:16:49

单位：200032 上海医科大学医学分子病毒学实验室(兰水云 袁正宏 闻玉梅)，上海市传染病医院肝炎免疫室(胡芸文，蒋伟伦)

关键词: 丙型肝炎病毒；3′端非编码区；核苷酸序列；逆转录聚合酶链反应 【摘要】 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HCV 3′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HCV RNA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HCV RNA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 3′noncoding region of Chinese hepatitis C virus genome LAN Shuiyun, YUAN Zhenghong, HU Yunwen, et al. Department of Molecular Virology, Shanghai Medical University, Shanghai 200032 【Abstract】 Objective To analyze the 3′ noncoding region (3′NCR) 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 full-length of the 3′ noncoding region of HCV genome from sera of HCV infected patients in Shanghai area: one was to amplify the full-length 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 3′NCR based RT-PCR was developed and its specificity and sensitivity for HCV RNA detection in sera was compared with the established 5′NCR based RT-PCR.Results Sequence analysis showed that Chinese HCV genomic 3′ NCR consists of three parts: the 5′ region, poly (U-UC) tract and the 98-base region. Sequence alignments revealed that, while the 98-base regions were completely conserved in different isolates and were identical to the reported sequences, the poly (U-UC) region shared highly diversities. A high degree of concordance(95%) between the 3′NCR and 5′NCR RT-PCR 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 3′NCR based RT-PCR may be a useful addition to available systems to diagnosis HCV infection. 【Key words】 Hepatitis C virus 3′ noncoding region Nucleotide sequence Reverse transcription polymerase chain reaction 自1989年美国学者发现丙型肝炎病毒(HCV)部分基因序列以来，已有许多HCV全基因组序列、研究编码基因结构与功能的报道^［1，2］。现已知，HCV基因组为单股正链RNA，全长约9.6 kb，由5′端非编码区(5′NCR)、编码3 000多个氨基酸的长阅读框架及3′端非编码区组成(3′NCR)。与5′NCR基因的长度及序列相对保守(319～341)核苷酸，同源性大于90%)相比，3′NCR的长度及序列在不同报道则有较大差异^［3，4］。本研究根据国外报道及中国人HCV基因组已知序列^［5］的特点设计引物，对中国人抗HCV阳性血清作RT-PCR，获得了中国人HCV基因组3′NCR的特异性PCR产物。克隆、测序分析发现，中国株HCV基因组3′NCR由高度变异区、Poly(U)区、Poly(U/C)区和98碱基保守区4部分组成；98碱基区与国外报道序列一致，其他结构在不同分离株间存在较大差异。3′NCR中，98碱基区在不同地区分离株间的高度保守，支持其在HCV基因组复制中可能具有重要作用。在此基础上，本研究建立了针对HCV基因3′NCR的RT-PCR方法。与5′NCR的RT-PCR同时检测42份抗HCV阳性及阴性血清发现，两者有较高的符合率(95%)。在抗体阳性血清中，前者的检出率略大于后者(71.8%，65.6%)。 1 材料和方法

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