
我国丁型肝炎病毒全基因克隆及序列分析
Cloning and sequence analysis of full-length cDNA of hepatitis D virus in China
为构建我国丁型肝炎病毒(HDV)全基因克隆,分别从3份HDV血清学阳性血清中提取病毒RNA,通过反转录-聚合酶链反应(RT-PCR)分段扩增,获得4个相互重叠的DNA片段,将PCR产物测序,利用DNAStar软件拼接,获得HDV全基因组序列;设计引物,用重叠PCR扩增全长HDV片段并连接至克隆载体,构建全基因克隆。结果成功克隆出3株1 675 bp的HDV全长基因组。经与GenBank标准序列比对,3株HDV均为基因Ⅰb型,核酸序列同源性达98%以上,与我国Ⅰ型X77627的同源性均达98%以上,与其他基因Ⅰ型的同源性均>84%。与基因Ⅱ型和Ⅲ型的同源性分别低于77%和65%。本研究构建的3株具有我国代表性的HDV全长基因cDNA克隆,为进一步开展HDV分子生物学研究提供了基础。
The present paper aims to clone and analyze the sequence of full-length cDNA of hepatitis D virus (HDV) in China. HDV RNA was extracted from three HDV seropositive samples. cDNA was gotten by reverse transcription-polymerase chain reaction (RT-PCR). Four overlapped fragments were amplified by nested PCR, and the products were sequenced. The full-length sequences were joined by DNAStar. The full-length cDNA of HDV was amplified by overlapping PCR using designed primers and connected to the cloning T vector. Three strains of HDV were successfully amplified and cloned. The three HDV strains were all genotype I. The nucleic acid sequence homology of the three HDV strains was over 98%. The nucleic acid molecules of three strains had at least 84% sequence homology to genotype Ib. But they had over 98% sequence homology to genotype I Chinese strain X77627. They had no more than 77% and 65% sequence homology compared with genotype II and genotype III respectively. In conclusion, we cloned full-length cDNA of three strains of HDV, which could be helpful for the further study of HDV molecular biology.
Hepatitis D virus / Polymerase chain reaction / Full-length genome
“十二五”国家重大传染病防治科技重大专项(2012ZX10004701)
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